Broadly cross-reactive neutralizing antibodies against human immunodeficiency virus selected by env-cd4-co-receptor complexes

ABSTRACT

The present invention features antibodies and antibody fragments that specifically bind a CD4-inducible HIV gp120 epitope that is enhanced by binding a co-receptor for HIV, such as CCR5 or CXCR4, and pharmaceutical compositions comprising the antibodies or antibody fragments. The invention also features nucleic acids encoding the antibodies or antibody fragments, pharmaceutical compositions comprising the nucleic acids encoding the antibodies or antibody fragments, vectors comprising the nucleic acids, and cells comprising the vectors. The invention further features methods of identifying antibodies or antibody fragments with broadly neutralizing activity against HIV. The invention also features methods of inhibiting HIV entry into cells and methods of inhibiting replication of HIV in mammals, using the antibodies and nucleic acids of the invention.

STATEMENT OF FEDERALLY SPONSORED RESEARCH

[0001] This invention was made with government support under ContractNo. AI33292 by the National Institutes of Health. The government of theUnited States of America has certain rights in the invention.

FIELD OF THE INVENTION

[0002] This invention generally relates to broadly neutralizingantibodies against Human Immunodeficiency Virus, and methods of makingand using the same.

BACKGROUND OF THE INVENTION

[0003] The Human Immunodeficiency Virus (HIV) is the causative agent ofAcquired Immunodeficiency Syndrome (AIDS). HIV entry into cells involvesformation of a complex between the HIV envelope glycoprotein (Env, whichconsists of a complex containing the HIV glycoproteins gp120 and gp41;gp120-gp41), a cell-surface receptor (CD4), and a cell-surfaceco-receptor (e.g., the chemokine receptor CCR5 or CXCR4). Binding of Envto CD4 and either co-receptor initiates a series of conformationalchanges that are the heart of the fusion machinery leading to viralentry into the target cell. Therefore, efforts to develop a vaccine forthe prevention and/or treatment of HIV infection have focused upon thedevelopment of neutralizing antibodies that specifically bind to Env.However, the extensive variation of Env in the numerous isolates of HIVso far identified presents a major obstacle in designing an effectiveimmunogen for the isolation of antibodies with broadly neutralizingactivity against multiple HIV isolates.

[0004] Currently there are only three well-characterized monoclonalantibodies (mAbs) with broadly neutralizing activity: the anti-gp120mAbs b12 (Burton et al. Science 266:1024-1027, 1994) and 2G12 (Trkola etal. J. Virol. 70:1100-1108, 1996), and the anti-gp41 mAb 2F5 (Conley etal. Proc. Natl. Acad. Sci. U. S. A. 91:3348-3352, 1994). Given theever-increasing number of people infected with HIV, there is a need inthe art for additional antibodies with broadly neutralizing activityagainst HIV, which can be used as passive immunotherapy or passiveimmunoprophylaxis to treat, ameliorate, inhibit, or prevent HIVinfections in individuals who have, or who at risk for developing, suchinfections. Furthermore, there is a need in the art for new strategiesby which to identify and/or isolate such broadly neutralizing anti-HIVantibodies.

SUMMARY OF THE INVENTION

[0005] We have discovered that purified complexes containing HIV Envtogether with the cell-surface HIV receptor CD4 and an HIV co-receptor,e.g., CCR5 or CXCR4, can be used to identify and isolate antibodies, andactive fragments thereof, which display broadly neutralizing activityagainst multiple genetic subtypes of HIV. Such antibodies can be used asinhibitors of HIV infection and for development of HIV vaccines.

[0006] In a first aspect, the invention relates to an isolated antibodyor antibody fragment that specifically binds a CD4-inducible epitope onHuman Immunodeficiency Virus (HIV) Env that is enhanced by the bindingof Env to a co-receptor for HIV, wherein the CD4-inducible epitope isdistinct from the HIV co-receptor binding site on gp120.

[0007] In one embodiment of the first aspect of the invention, theantibody or antibody fragment is selected by virtue of its ability tospecifically bind to a CD4-inducible epitope on HIV Env that is enhancedby binding a co-receptor for HIV.

[0008] In a second aspect, the invention relates to an isolated antibodyor antibody fragment that is selected by virtue of its ability tospecifically bind to a CD4-inducible epitope on Human ImmunodeficiencyVirus (HIV) Env that is enhanced by the binding of Env to a co-receptorfor HIV, and wherein the CD4-inducible epitope is distinct from the HIVco-receptor binding site on gp120.

[0009] In various embodiments of the first and second aspects of theinvention, the epitope can be on gp120, on gp41, or on gp120-gp41 (Env).

[0010] In a third aspect, the invention relates to an isolated antibodyor antibody fragment that is selected by virtue of its ability tospecifically bind to a complex comprising HIV gp120, CD4, and aco-receptor for HIV. In one embodiment of the third aspect of theinvention, the complex also comprises gp41.

[0011] In various embodiments of the first, second, and third aspects ofthe invention, the HIV co-receptor can be CCR5 or CXCR4; the isolatedantibody or antibody fragment can have broadly neutralizing activityagainst HIV, e.g., HIV-1; the isolated antibody or antibody fragment canbe monoclonal; the isolated antibody or antibody fragment can be humanor humanized; and/or the isolated antibody or antibody fragment can beisolated from a phage display library.

[0012] In other embodiments of the first, second, and third aspects ofthe invention, the antibody or antibody fragment can comprise the heavychain of the Fab fragment X5 (SEQ ID NO: 3), the light chain of the Fabfragment X5 (SEQ ID NO: 2), or both chains of X5.

[0013] In still other embodiments of the first, second, and thirdaspects of the invention, the isolated antibody or antibody fragment cancomprise the CDR3 region (SEQ ID NO: 5) of the heavy chain of the Fabfragment X5 and/or the CDR3 region (SEQ ID NO: 8) of the light chain ofthe Fab fragment X5. The antibody or antibody fragment can also compriseany of the other CDR and/or FR regions found in the heavy or light chainof antibody Fab fragment X5, in any combination.

[0014] In yet other embodiments of the first, second, and third aspectsof the invention, a fusion polypeptide comprising a heavy chain or lightchain of the antibody or antibody fragment can comprise a soluble CD4(sCD4) domain. Such a polypeptide can further comprise an amino acidsequence corresponding to that of the peptide T20, which is a syntheticpeptide derived from the HIV gp41 amino acid sequence.

[0015] In a fourth aspect, the invention relates to an isolatedpolypeptide comprising the heavy chain of antibody Fab fragment X5 (SEQID NO: 3).

[0016] In a fifth aspect, the invention relates to an isolatedpolypeptide comprising the light chain of antibody Fab fragment X5 (SEQID NO: 2).

[0017] In a sixth aspect, the invention relates to an isolatedpolypeptide comprising the CDR3 region (SEQ ID NO: 5) of the heavy chainof antibody Fab fragment X5.

[0018] In a seventh aspect, the invention relates to an isolatedpolypeptide comprising the CDR3 region (SEQ ID NO: 8) of the light chainof antibody Fab fragment X5.

[0019] In an eighth aspect, the invention relates to an isolatedpolypeptide comprising the CDR3 region (SEQ ID NO: 5) of the heavy chainof antibody Fab fragment X5 and the CDR3 region (SEQ ID NO: 8) of thelight chain of antibody Fab fragment X5. For example, the polypeptidecan be a single chain antibody or a single chain antibody fragment, suchas a single chain variable fragment (ScFv).

[0020] In a ninth aspect, the invention relates to an antibody orantibody fragment that is an amino acid sequence variant of the Fabfragment X5, wherein the sequence variant of X5 comprises at least oneamino acid substitution in the heavy chain or light chain of X5, whereinthe sequence variant of X5 binds a complex comprising gp120, CD4, and anHIV-co-receptor with an affinity that is about equal to or greater thanthe affinity by which X5 binds the comprising gp120, CD4, and anHIV-co-receptor.

[0021] In one embodiment of the ninth aspect of the invention, thesequence variant of X5 has broadly neutralizing activity against HIV-1.In other embodiments, the amino acid substitution is in the CDR3 regionof the heavy chain and/or light chain of X5. The amino acid substitutioncan also be in any other region of the heavy or light chains, e.g., inany of the CDR, FR, or CH1 regions; for example, the amino acidsubstitution can be in CH1, and the sequence variant of X5 can compriseSEQ ID NO: 11.

[0022] In another embodiment of the ninth aspect of the invention, thesequence variant of X5 is selected by virtue of its ability tospecifically bind to a complex comprising HIV gp120, CD4, and aco-receptor for HIV. In another embodiment, the sequence variant of X5is selected by virtue of its ability to specifically bind to aCD4-inducible epitope on HIV Env that is enhanced by binding aco-receptor for HIV.

[0023] In a tenth aspect, the invention relates to an isolatedpolypeptide comprising an amino acid sequence variant of a CDR3 regionof antibody Fab fragment X5, wherein an antibody or antibody fragmentcomprising the amino acid sequence variant of the CDR3 region of X5binds HIV gp120 with an affinity that is about equal to or greater thanto the affinity by which X5 binds gp120. The CDR3 region may be from theheavy chain or the light chain of antibody X5.

[0024] In one embodiment of the ninth and tenth aspects of theinvention, the amino acid sequence variant is selected by virtue of itsequivalent or increased affinity for gp120 relative to the affinity ofX5 for gp120.

[0025] In an eleventh aspect, the invention relates to an isolatednucleic acid that encodes SEQ ID NO: 3.

[0026] In a twelfth aspect, the invention relates to an isolated nucleicacid that encodes SEQ ID NO: 2.

[0027] In a thirteenth aspect, the invention relates to an isolatednucleic acid that encodes SEQ ID NO: 5.

[0028] In a fourteenth aspect, the invention relates to an isolatednucleic acid that encodes SEQ ID NO: 8.

[0029] In a fifteenth aspect, the invention relates to an isolatednucleic acid that encodes an antibody or antibody fragment comprisingthe CDR3 region (SEQ ID NO: 5) of the heavy chain of antibody Fabfragment X5 and the CDR3 region (SEQ ID. NO: 8) of the light chain ofantibody Fab fragment X5.

[0030] In a sixteenth aspect, the invention relates to an isolatednucleic acid that encodes an antibody or antibody fragment comprisingthe heavy chain of antibody Fab fragment X5 (SEQ ID NO: 3) and the lightchain of antibody Fab fragment X5 (SEQ ID NO: 2). For example, theisolated nucleic acid can comprise the nucleotide sequence set forth inSEQ ID NO: 4.

[0031] In a seventeenth aspect, the invention relates to an isolatedvector comprising the isolated nucleic acid of aspects eleven throughsixteen above. The vector can be, for example, an expression vector forexpression of the peptide or polypeptide encoded by the isolated nucleicacid.

[0032] In an eighteenth aspect, the invention relates to an isolatedcell comprising the isolated vector of the seventeenth aspect of theinvention. The cell can be a prokaryotic cell or a eukaryotic cell.

[0033] In a nineteenth aspect, the invention relates to a pharmaceuticalcomposition comprising the isolated antibody or antibody fragment of thefirst three aspects and the ninth aspect of the invention, and apharmaceutically acceptable carrier. The pharmaceutical composition canfurther comprise soluble CD4.

[0034] In a twentieth aspect, the invention relates to a pharmaceuticalcomposition comprising a nucleic acid that encodes the isolated antibodyor antibody fragment of the first three aspects and the ninth aspect ofthe invention, and a pharmaceutically acceptable carrier. In oneembodiment of the twentieth aspect of the invention, the nucleic acid iswithin an expression vector.

[0035] In a twenty-first aspect, the invention relates to a method ofselecting an antibody or antibody fragment with broadly neutralizingactivity against HIV, comprising detecting an antibody or antibodyfragment that specifically binds a CD4-inducible epitope on HIV Env thatis enhanced by the binding of Env to a co-receptor for HIV, wherein theCD4-inducible epitope is distinct from the HIV co-receptor binding siteon gp120. For example, the antibody or antibody fragment can be selectedby virtue of its binding to a complex comprising HIV gp120, CD4, and aco-receptor for HIV.

[0036] In a twenty-second aspect, the invention relates to an antibodyproduced by the method of the twenty-first aspect of the invention.

[0037] In a twenty-third aspect, the invention relates to a method ofinhibiting entry of HIV into a cell, comprising administering to thecell an effective amount of an isolated antibody or antibody fragmentthat specifically binds a CD4-inducible epitope on HIV Env that isenhanced by the binding of Env to a co-receptor for HIV, wherein theCD4-inducible epitope is distinct from the HIV co-receptor binding siteon gp120, thereby inhibiting entry of HIV into the cell.

[0038] In various embodiments of the twenty-third aspect of theinvention, the cell can be any cell susceptible to HIV infection, e.g.,but not limited to, a T cell, a B cell, a monocyte, a macrophage, or amicroglial cell. In another embodiment of the twenty-third aspect of theinvention, the cell is within a mammal that is susceptible to infectionby HIV and the isolated antibody or antibody fragment is administered tothe mammal.

[0039] In a twenty-fourth aspect, the invention relates to a method ofinhibiting replication of HIV in a mammal that is susceptible to HIVinfection, comprising administering to the mammal an effective amount ofan isolated antibody or antibody fragment that specifically binds aCD4-inducible epitope on HIV Env that is enhanced by the binding of Envto a co-receptor for HIV, wherein the CD4-inducible epitope is distinctfrom the HIV co-receptor binding site on gp120, thereby inhibitingreplication of HIV in the mammal.

[0040] In various embodiments of the twenty-third and twenty-fourthaspects of the invention, the isolated antibody or antibody fragment isadministered to the mammal by administering a nucleic acid encoding theisolated antibody or antibody fragment to the mammal.

[0041] In other embodiments of the twenty-third and twenty-fourthaspects of the invention the mammal is a primate, for example, a humanor a non-human primate.

[0042] In all of the above embodiments of the invention, the HIV can beHIV-1 or HIV-2.

[0043] In all of the above embodiments of the invention, the co-receptorcan be, e.g., CCR5 or CXCR4.

[0044] Additional advantages of the invention will be set forth in partin the description which follows, and in part will be apparent from thedescription, or may be learned by practice of the invention. Theadvantages of the invention will be realized and attained by means ofthe elements and combinations particularly pointed out in the appendedclaims. It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory only and are not restrictive of the invention as claimed.

[0045] Definitions

[0046] In this specification and in the claims which follow, referencewill be made to a number of terms which shall be defined to have thefollowing meanings. It is to be understood that the terminology usedherein is for the purpose of describing particular embodiments only andis not intended to be limiting.

[0047] By “specifically binds”, “specifically reacts with”,“specifically interacts with”, and similar terms is meant that ananti-HIV antibody of the invention physically associates with its targetmolecule (e.g., gp120 and/or gp41 of HIV Env) to inhibit HIV entry intoa cell and/or to inhibit or prevent HIV replication in a mammal.Preferably, the antibody does not substantially physically associatewith other molecules.

[0048] By a “broadly neutralizing” antibody against HIV, and similarterms, is meant an antibody that can inhibit the activity (e.g., theability to enter a target cell) of HIV isolates from more than onegenetic subtype or clade.

[0049] By “CD4-inducible epitope on HIV Env that is distinct from theco-receptor binding site on gp120” is meant that an antibody of theinvention does not compete with an HIV co-receptor (e.g., CCR5 or CXCR4)for the co-receptor binding site on gp120. One of ordinary skill in theart will understand how to determine whether an antibody competes with aco-receptor for the co-receptor binding site on Env, using well-knowntechniques for measuring competition between two molecules for bindingto a particular site on a third molecule.

[0050] By “selected” is meant that an antibody or antibody fragment ofthe invention is chosen or isolated from a group or library of candidateantibodies or antibody fragments using a screening assay for choosing orisolating antibodies with a desired characteristic (e.g., the ability tobind a complex comprising HIV gp120, CD4, and a co-receptor for HIV; orthe ability to specifically bind a CD4-inducible epitope on HIV Env thatis enhanced by the binding of Env to a co-receptor for HIV, wherein theCD4-inducible epitope is distinct from the HIV co-receptor binding siteon gp120), as would be understood by one of ordinary skill in the art.

[0051] By “CD4-inducible epitope” is meant an antigenic site on HIV Env,gp120, or gp 41, wherein specific binding to the antigenic site by anantibody of the invention is increased or augmented by the binding ofCD4 to HIV Env, gp120, or gp41. Preferably the increase is by at leastabout 2-fold or greater, e.g., at least about: 2-fold, 3-fold, 4-fold,5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more. For example,the binding of the anti-HIV antibody X5 (described herein) to gp120 isincreased by about 5- to 10-fold when CD4 is present (i.e., when gp120is bound to CD4, e.g., sCD4).

[0052] By “exposure of the epitope is enhanced” or “epitope that isenhanced” is meant that specific binding of an antibody of the inventionto its cognate CD4-inducible epitope on HIV Env, gp120, or gp41 isfurther augmented by the binding of HIV Env, gp120, or gp41 to aco-receptor for HIV (such as the chemokine receptors CCR5 or CXCR4).Preferably the increase is by at least about 1.2-fold or greater, e.g.,at least about 1.3-, 1.4-, 1.5-, 1.6-, 1.7-, 1.8-, 1.9-, or 2-fold, orgreater.

[0053] By “soluble CD4” or “sCD4” or D1D2” is meant a CD4 molecule, or afragment thereof, that is in aqueous solution and that can mimic theactivity of native membrane-anchored CD4 by altering the conformation ofHIV Env, as is understood by those of ordinary skill in the art. Oneexample of a soluble CD4 is the two-domain soluble CD4 (sCD4 or D1D2)described, e.g., in Salzwedel et al. J. Virol. 74:326-333, 2000.

[0054] By “isolated polypeptide” is meant a polypeptide (or a fragmentthereof) that has been separated from the components that naturallyaccompany it. Typically, the polypeptide is substantially pure when itis at least 60%, by weight, free from the proteins andnaturally-occurring organic molecules with which it is naturallyassociated. Preferably, the polypeptide is at least 75%, more preferablyat least 80% or 90%, and most preferably at least 95%, by weight, pure.A substantially pure polypeptide may be obtained, for example, byextraction from a natural source (e.g., a cell), by expression of arecombinant nucleic acid encoding the polypeptide, or by chemicallysynthesizing the polypeptide. Purity can be measured by any appropriatemethod, e.g., by column chromatography, polyacrylamide gelelectrophoresis, or HPLC analysis.

[0055] A protein is substantially free of naturally associatedcomponents when it is separated from those contaminants which accompanyit in its natural state. Thus, a protein which is chemically synthesizedor produced in a cellular system different from the cell from which itnaturally originates will be substantially free from its naturallyassociated components. Accordingly, substantially pure polypeptides notonly includes those derived from eukaryotic organisms but also thosesynthesized in E. coli or other prokaryotes.

[0056] By “isolated nucleic acid” is meant a nucleic acid molecule thatis free of the genes which, in the naturally-occurring genome of theorganism from which the DNA of the invention is derived, flank the gene.The term therefore includes, for example, a recombinant DNA which isincorporated into a vector; into an autonomously replicating plasmid orvirus; or into the genomic DNA of a prokaryote or eukaryote; or whichexists as a separate molecule (e.g., a cDNA or a genomic or cDNAfragment produced by PCR or restriction endonuclease digestion, or anmRNA transcribed from a recombinant DNA template) independent of othersequences. It also includes a recombinant DNA which is part of a hybridgene encoding additional polypeptide sequence.

[0057] By “expression vector” is meant a DNA construct that contains apromoter operably linked to a downstream gene or coding region (e.g., acDNA or genomic DNA fragment, which encodes a polypeptide or polypeptidefragment). Introduction of the expression vector into the appropriaterecipient cell (e.g., a prokaryotic or eukaryotic cell, e.g., abacterium, yeast, insect cell, or mammalian cell, depending upon thepromoter within the expression vector) allows the cell to express mRNAencoded by the expression vector, which is then translated into theencoded polypeptide or polypeptide fragment. Vectors for in vitrotranscription/translation are also well-known in the art. An expressionvector may be a genetically engineered plasmid, virus, or artificialchromosome derived from, for example, a bacteriophage, adenovirus,retrovirus, poxvirus, or herpesvirus.

[0058] By “effective amount” is meant the amount of an anti-HIV antibodyof the invention that is useful for treating, partially or completelyinhibiting, or preventing an HIV infection in a patient or subject orpartially or completely inhibiting entry of HIV into a cell, asdescribed herein. Effective dosages and schedules for administering theantibodies of the invention may be determined empirically, and makingsuch determinations is routine to one of ordinary skill in the art. Theskilled artisan will understand that the dosage of anti-HIV antibodieswill vary, depending upon, for example, the species of the subject theroute of administration, the particular antibody to be used, other drugsbeing administered, and the age, condition, sex and extent of thedisease in the subject. The dosage can be adjusted by the individualphysician in the event of any counter-indications. A effective dose ofan anti-HIV antibody of the invention generally will range between about1 μg/kg of body weight and 100 mg/kg of body weight. Examples of suchdosage ranges are (but are not limited to), e.g., about 1 μg-100 μg/kg100 μg-1 mg/kg, 1 mg/kg-10 mg/kg, or 10 mg-100 mg/kg, once a week,bi-weekly, daily, or two to four times daily.

DESCRIPTION OF THE DRAWINGS

[0059]FIG. 1 is a bar graph showing that the X5 epitope is induced bysCD4 and its exposure enhanced by dsCCR5. BSA, gp120_(JRFL),gp120_(JRFL)+sCD4, gp120_(Bal)-CD4, sCD4 (D1D2) or dsCCR5 (100 ng eachin 0.1 ml solution) were coated on an ELISA plate overnight at 4° C.after which the wells were blocked with 4% milk in Tris-buffered saline.Detergent (0.5% Cymal-5)-solubilized CCR5 (dsCCR5) at 100 ng per wellcontaining 0.1 ml buffer was added. In control experiment without dsCCR5Cymal-5 (0.5%) buffer was added and incubated overnight at 4° C., thenX5 Fab was used as primary antibody (Ab) followed by goat anti-humanF(ab′)₂-HRP to detect the signal.

[0060]FIG. 2 is a diagram showing binding isotherms of X5 to gp120 andgp120-sCD4. Experimental data for gp120_(89.6) and sCD4-gp120_(89.6)obtained as described for FIG. 1 were analyzed by fitting them to anequation describing the Langmuir adsorption isotherm(B/B_(max)=X5/(K_(d)+X5), where B is the amount of bound X5, B_(max) isthe maximal amount of bound X5, X5 is its bulk concentration and K_(d)is the equilibrium dissociation constant (affinity is the inverse ofK_(d)). The continuous lines represent fitting of the data for X5binding to gp120_(89.6) (lower curve) and sCD4-gp120_(89.6) (uppercurve) with K_(d) equal to 9.3 nM and 1.0 nM, respectively.

[0061]FIG. 3 is a graph showing binding of antibodies X5, b12, and 17bto cell surface-associated functional oligomeric gp120-gp41. The T-cellline H9 was infected with the HIV-1 TCLA X4 MN isolate for 10 days. Atthis time post-infection there was no detectable CD4 remaining at thecell surface, and no syncytium formation in the culture, but strong Envexpression was detected using gp120-specific mAbs and flow cytometry. H9cells were pre-incubated with sCD4 (20 μg/ml) or buffer alone for 1 h atRT, then incubated with X5 Fab, the anti-CD4i mAb 17b or the anti-CD4BSFab IgG1b12 at various concentrations. The amount of bound antibodieswas measured by flow cytometry.

[0062]FIG. 4 is a bar graph showing inhibition of sCD4-induced fusion byX5. Fusion between 293 cells, expressing CCR5 or CXCR4 after infectionwith recombinant vaccinia viruses (PM1107 or DM1107, respectively), and293 cells, expressing HIV-1_(89.6)Env after infection with recombinantvaccinia virus, induced by soluble CD4, was measured by thebeta-galactosidase assay. The Env-expressing cells were mixed with sCD4(1 ug/ml) and X5 (0, 125, 250, 500 ng/ml) for 30 min at 37° C. In acontrol experiment (con) no sCD4 was added. These cells were mixed withthe cells expressing receptor molecules at a ratio of 1:1 (total numberof cells equal to 2×105 in 96-well plate format). Fusion was allowed toproceed for 2 h at 37° C. and quantitated by a colorimetric assay whichmeasures the optical density at 595 nm (OD₅₉₅).

[0063]FIGS. 5A and 5B are graphs showing neutralization of HIV infectionby X5. Infection of HOS CD4.CXCR4 or HOS CD4.CCR5 cells by pseudotypedHIV-1_(NL4-3) was monitored by a reporter gene assay performed asdescribed in the Materials and Methods section of Example I.

[0064]FIGS. 6A and 6B are graphs showing antibody competition assays forcharacterization of the X5 epitope. To characterize the X5 epitope,known CD4-inducible monoclonal antibodies (mAbs) were tested in ancompetition ELISA assay. Nunc-Immuno™ Maxisorp™ surface plates (NalgeNunc International, Denmark) were coated with 100 ul of gp120 (FIG. 6A)or gp120+CD4 (FIG. 6B) (0.5 ug/ml each) in carbonated buffer, blockedwith 4% milk in TBS. Fab X5 was biotinylated and a fixed concentration(0.5 ug/ml) was added to each well along with increasing concentrations(0, 0.01, 0.1, 1, 10 ug/ml) of indicated mAbs. Binding of biotinylatedX5 was detected using streptavidin-HRP secondary Ab. Unbiotinylated X5was also tested (at 0, 0.01, 0.1, 1, and 10 ug/ml) and more than 50%inhibition was detected at 1 ug/ml concentration.

[0065]FIG. 7 is a diagram showing the sequence of the X5 light chain(SEQ ID NO: 2). The OmpA signal sequence, three framework regions(FR1-FR3), three complementarity-determining regions (CDR1-CDR3), kappachain joining region (Jκ), and light chain constant region (CL) areindicated by heavy horizontal lines above the relevant amino acids.

[0066]FIG. 8 is a diagram showing the sequence of the X5 heavy chain(SEQ ID NO: 3). The PelB signal sequence, four framework regions(FR1-FR4), three complementarity-determining regions (CDR1-CDR3), andheavy chain constant region 1 (CH1) are indicated by heavy horizontallines above the relevant amino acids. The two amino acid positions thatare substituted in the X5 sequence variant FabS (described in ExampleII), are shown below the original X5 heavy chain sequence.

DETAILED DESCRIPTION OF THE INVENTION

[0067] Binding of the HIV envelope glycoprotein (also known as Env orgp120-gp41) to CD4 and the co-receptor CCR5 or CXCR4 initiates a seriesof conformational changes that are the heart of the fusion machineryleading to viral entry. The elucidation of the nature of the Envconformational changes is not only a clue to the mechanism of HIV-1entry but also provides new tools for the development of inhibitors andvaccines.

[0068] Described herein is a novel approach for the identification ofbroadly cross-reactive antibodies that neutralize multiple geneticsubtypes of HIV. This approach involves the use of purifiedEnv-CD4-co-receptor complexes for screening libraries of antibodies orantibody fragments that specifically bind to receptor-inducible HIVepitopes. Such antibodies can be used for treating, inhibiting, and/orpreventing HIV infection by providing passive immunity to treatedindividuals. Currently there are known only three well characterizedmonoclonal antibodies with broadly neutralizing activity, and none ofthese antibodies is directed against a receptor-inducible epitope.

[0069] Using this approach, a novel human antibody Fab fragment, denoted“X5”, was identified by screening a phage display library, as describedin Example I below. The epitope recognized by the X5 antibody isinducible by CD4 and exposure of the epitope is enhanced by the majorHIV-1 co-receptor CCR5. The antibody neutralizes R5 and R5X4 viruses,including primary isolates, and to lesser extent, X4 viruses. Sequencevariants and antibody fusion proteins based on X5 are also describedherein, and will be apparent to those of ordinary skill in the art.

[0070] Antibodies

[0071] The term “antibodies” is used herein in a broad sense andincludes both polyclonal and monoclonal antibodies. In addition tointact immunoglobulin molecules, also included in the term “antibodies”are fragments of immunoglobulin molecules and multimers ofimmunoglobulin molecules (e.g., diabodies, triabodies, and bi-specificand tri-specific antibodies, as are known in the art; see, e.g., Hudsonand Kortt, J. Immunol. Methods 231:177-189, 1999), fusion proteinscontaining an antibody or antibody fragment (e.g., a fusion proteincontaining a fragment of CD4, e.g., sCD4 (Salzwedel et al. J. Virol.74:326-333, 2000), which are produced using standard molecular biologytechniques, single chain antibodies, and human or humanized versions ofimmunoglobulin molecules or fragments thereof, as long as they arechosen for their ability to broadly neutralize HIV (e.g., multiplegenetic subtypes of HIV-1 or HIV-2) by binding a CD4-inducible HIVepitope that is enhanced by binding an HIV co-receptor, as describedherein. The antibodies are tested for their desired activity using thein vitro assays described herein, or by analogous methods, after whichtheir in vivo therapeutic and/or prophylactic activities are testedaccording to known clinical testing methods.

[0072] The term “monoclonal antibody” as used herein refers to anantibody or antibody fragment obtained from a substantially homogeneouspopulation of antibodies or antibody fragments, i.e., the individualantibodies within the population are identical except for possiblenaturally occurring mutations that may be present in a small subset ofthe antibody molecules. The monoclonal antibodies herein specificallyinclude “chimeric” antibodies in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, as long asthey exhibit the desired antagonistic activity (See U.S. Pat. No.4,816,567 and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855(1984)).

[0073] Monoclonal antibodies of the invention can be prepared usinghybridoma methods, such as those described by Kohler and Milstein,Nature, 256:495 (1975). In a hybridoma method, a mouse or otherappropriate host animal is typically immunized with an immunizing agentto elicit lymphocytes that produce or are capable of producingantibodies that will specifically bind to the immunizing agent.Alternatively, the lymphocytes may be immunized in vitro, e.g., usingthe HIV Env-CD4-co-receptor complexes described herein.

[0074] The monoclonal antibodies may also be made by recombinant DNAmethods, such as those described in U.S. Pat. No. 4,816,567 (Cabilly etal.). DNA encoding the monoclonal antibodies of the invention can bereadily isolated and sequenced using conventional procedures (e.g., byusing oligonucleotide probes that are capable of binding specifically togenes encoding the heavy and light chains of murine antibodies).Libraries of antibodies or active antibody fragments can also begenerated and screened using phage display techniques, e.g., asdescribed in U.S. Pat. No. 5,804,440 (Burton et al.) and U.S. Pat. No.6,096,441 (Barbas et al.). Recombinant antibodies, antibody fragments,and fusions and polymers thereof can be expressed in vitro or inprokaryotic cells (e.g., bacteria) or eukaryotic cells (e.g., yeast,insect, or mammalian cells) and further purified, as necessary, usingwell known methods (see, e.g., Sambrook et al. Molecular Cloning: aLaboratory Manual, Cold Spring Harbor Laboratory Press (1989); andAusubel et al., Current Protocols in Molecular Biology, John Wiley &Sons, New York, N.Y., 2001, which is updated quarterly).

[0075] In vitro methods are also suitable for preparing monovalentantibodies. Digestion of antibodies to produce fragments thereof,particularly, Fab fragments, can be accomplished using routinetechniques known in the art. For instance, digestion can be performedusing papain. Examples of papain digestion are described in WO 94/29348published Dec. 22, 1994 and U.S. Pat. No. 4,342,566. Papain digestion ofantibodies typically produces two identical antigen binding fragments,called Fab fragments, each with a single antigen binding site, and aresidual Fc fragment. Pepsin treatment yields a fragment that has twoantigen combining sites and is still capable of cross-linking antigen.

[0076] Any antibody or antibody fragment of the invention, whetherattached to other sequences or not, can also include insertions,deletions, substitutions, or other selected modifications of particularregions or specific amino acids residues, provided the activity of theantibody or antibody fragment is not significantly altered or impairedcompared to the non-modified antibody or antibody fragment. Thesemodifications can provide for some additional property, such as toremove/add amino acids capable of disulfide bonding, to increase itsbio-longevity, to alter its secretory characteristics, etc. In any case,the antibody or antibody fragment must possess a bioactive property,such as specific binding to its cognate antigen. Functional or activeregions of the antibody or antibody fragment may be identified and/orimproved by mutagenesis of a specific region of the protein, followed byexpression and testing of the expressed polypeptide. For example, aminoacid sequence variants of antibodies or antibody fragments can begenerated and those that display equivalent or improved affinity forantigen can be identified using standard techniques and/or thosedescribed herein. Methods for generating amino acid sequence variantsare readily apparent to a skilled practitioner in the art and caninclude site-specific mutagenesis or random mutagenesis (e.g., by PCR)of the nucleic acid encoding the antibody or antibody fragment (Zoller,M. J. Curr. Opin. Biotechnol. 3:348-354, 1992). Both naturally occurringand non-naturally occurring amino acids (e.g., artificially-derivatizedamino acids) may be used to generate amino acid sequence variants of theantibodies and antibody fragments of the invention.

[0077] As used herein, the term “antibody” or “antibodies” can alsorefer to a human antibody and/or a humanized antibody. Many non-humanantibodies (e.g., those derived from mice, rats, or rabbits) arenaturally antigenic in humans, and thus can give rise to undesirableimmune responses when administered to humans. Therefore, the use ofhuman or humanized antibodies in the methods of the invention serves tolessen the chance that an antibody administered to a human will evoke anundesirable immune response.

[0078] Human Antibodies

[0079] The human antibodies of the invention can be prepared using anytechnique. Examples of techniques for human monoclonal antibodyproduction include those described by Cole et al. (Monoclonal Antibodiesand Cancer Therapy, Alan R. Liss, p. 77, 1985) and by Boerner et al. (J.Immunol., 147(1):86-95, 1991). Human antibodies of the invention (andfragments thereof) can also be produced using phage display libraries(Hoogenboom et al., J. Mol. Biol., 227:381, 1991; Marks et al., J. Mol.Biol., 222:581, 1991; and C. F. Barbas, D. R. Burton, J. K. Scott, G. J.Silverman, Phage Display: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 2001).

[0080] The human antibodies of the invention can also be obtained fromtransgenic animals. For example, transgenic, mutant mice that arecapable of producing a full repertoire of human antibodies, in responseto immunization, have been described (see, e.g., Jakobovits et al.,Proc. Natl. Acad. Sci. USA, 90:2551-255 (1993); Jakobovits et al.,Nature, 362:255-258 (1993); Bruggermann et al., Year in Immunol., 7:33(1993)). Specifically, the homozygous deletion of the antibody heavychain joining region (J(H)) gene in these chimeric and germ-line mutantmice results in complete inhibition of endogenous antibody production,and the successful transfer of the human germ-line antibody gene arrayinto such germ-line mutant mice results in the production of humanantibodies upon antigen challenge. Antibodies having the desiredactivity are selected using Env-CD4-co-receptor complexes as describedherein.

[0081] Humanized Antibodies

[0082] Antibody humanization techniques generally involve the use ofrecombinant DNA technology to manipulate the DNA sequence encoding oneor more polypeptide chains of an antibody molecule. Accordingly, ahumanized form of a non-human antibody (or a fragment thereof) is achimeric antibody or antibody chain (or a fragment thereof, such as anFv, Fab, Fab′, or other antigen-binding portion of an antibody) whichcontains a portion of an antigen binding site from a non-human (donor)antibody integrated into the framework of a human (recipient) antibody.

[0083] To generate a humanized antibody, residues from one or morecomplementarity determining regions (CDRs) of a recipient (human)antibody molecule are replaced by residues from one or more CDRs of adonor (non-human) antibody molecule that is known to have desiredantigen binding characteristics (e.g., a certain level of specificityand affinity for the target antigen). In some instances, Fv framework(FR) residues of the human antibody are replaced by correspondingnon-human residues. Humanized antibodies may also contain residues whichare found neither in the recipient antibody nor in the imported CDR orframework sequences. Generally, a humanized antibody has one or moreamino acid residues introduced into it from a source which is non-human.In practice, humanized antibodies are typically human antibodies inwhich some CDR residues and possibly some FR residues are substituted byresidues from analogous sites in rodent antibodies. Humanized antibodiesgenerally contain at least a portion of an antibody constant region(Fc), typically that of a human antibody (Jones et al., Nature,321:522-525 (1986), Reichmann et al., Nature, 332:323-327 (1988), andPresta, Curr. Opin. Struct. Biol., 2:593-596 (1992)).

[0084] Methods for humanizing non-human antibodies are well known in theart. For example, humanized antibodies can be generated according to themethods of Winter and co-workers (Jones et al., Nature, 321:522-525(1986), Riechmann et al., Nature, 332:323-327 (1988), Verhoeyen et al.,Science, 239:1534-1536 (1988)), by substituting rodent CDRs or CDRsequences for the corresponding sequences of a human antibody. Methodsthat can be used to produce humanized antibodies are also described inU.S. Pat. No. 4,816,567 (Cabilly et al.), U.S. Pat. No. 5,565,332(Hoogenboom et al.), U.S. Pat. No. 5,721,367 (Kay et al.), U.S. Pat. No.5,837,243 (Deo et al.), U.S. Pat. No. 5,939,598 (Kucherlapati et al.),U.S. Pat. No. 6,130,364 (Jakobovits et al.), and U.S. Pat. No. 6,180,377(Morgan et al.).

[0085] Administration of Antibodies

[0086] Antibodies of the invention are preferably administered to asubject in a pharmaceutically acceptable carrier. Suitable carriers andtheir formulations are described in Remington: The Science and Practiceof Pharmacy (19th ed.) ed. A. R. Gennaro, Mack Publishing Company,Easton, Pa. 1995. Typically, an appropriate amount of a pharmaceuticallyacceptable salt is used in the formulation to render the formulationisotonic. Examples of the pharmaceutically acceptable carrier include,but are not limited to, saline, Ringer's solution and dextrose solution.The pH of the solution is preferably from about 5 to about 8, and morepreferably from about 7 to about 7.5. Further carriers include sustainedrelease preparations such as semipermeable matrices of solid hydrophobicpolymers containing the antibody, which matrices are in the form ofshaped articles, e.g., films, liposomes or microparticles. It will beapparent to those persons skilled in the art that certain carriers maybe more preferable depending upon, for instance, the route ofadministration and concentration of antibody being administered.

[0087] The antibodies can be administered to the subject, patient, orcell by injection (e.g., but not limited to, intravenous,intraperitoneal, intradermal, subcutaneous, intramuscular), or by othermethods such as infusion that ensure its delivery to the bloodstream inan effective form. Local or intravenous injection is preferred.

[0088] Effective dosages and schedules for administering the antibodiesmay be determined empirically, and making such determinations is withinthe skill in the art. Those skilled in the art will understand that thedosage of antibodies that must be administered will vary depending on,for example, the subject that will receive the antibody, the route ofadministration, the particular type of antibody used and other drugsbeing administered. Guidance in selecting appropriate doses forantibodies is found in the literature on therapeutic uses of antibodies,e.g., Handbook of Monoclonal Antibodies, Ferrone et al., eds., NogesPublications, Park Ridge, N.J., (1985) ch. 22 and pp. 303-357; Smith etal., Antibodies in Human Diagnosis and Therapy, Haber et al., eds.,Raven Press, New York (1977) pp. 365-389. A typical daily dosage of theantibody used alone might range from about 1 μg/kg to up to 100 mg/kg ofbody weight or more per day, depending on the factors mentioned above.

[0089] Following administration of an antibody for treating, inhibiting,or preventing an HIV infection, the efficacy of the therapeutic antibodycan be assessed in various ways well known to the skilled practitioner.For instance, one of ordinary skill in the art will understand that anantibody of the invention is efficacious in treating or inhibiting anHIV infection in a subject by observing that the antibody reduces viralload or delays or prevents a further increase in viral load. Viral loadscan be measured by methods that are known in the art, for example, usingpolymerase chain reaction assays to detect the presence of HIV nucleicacid or antibody assays to detect the presence of HIV protein in asample (e.g., but not limited to, blood or another body fluid) from asubject or patient, or by measuring the level of circulating anti-HIVantibodies in the patient. Efficacy of the antibody treatment may alsobe determined by measuring the number of CD4+ T cells in theHIV-infected subject. An antibody treatment that delays or inhibits aninitial or further decrease in CD4+ T cells in an HIV-positive subjector patient, or that results in an increase in the number of CD4+ T cellsin the HIV-positive subject, is an efficacious antibody treatment.

[0090] The broadly-neutralizing antibodies of the invention can also beadministered prophylactically to patients or subjects who are at riskfor being exposed to HIV or who have been newly exposed to HIV. Suchpatients include, but are not limited to, healthcare workers; fetuses,neonates, or infants (e.g., nursing infants) whose mothers are infectedor at risk for being infected; intravenous drug users; recipients ofblood transfusions, blood products, or transplantation tissue; and otherindividuals who have been exposed to a body fluid that contains or maycontain HIV.

[0091] In subjects who have been newly exposed to HIV but who have notyet displayed the presence of the virus (as measured by PCR or otherassays for detecting the virus) in blood or other body fluid,efficacious treatment with an antibody of the invention partially orcompletely inhibits or delays the appearance of the virus or minimizesthe level of the virus in the blood or other body fluid of the exposedindividual.

[0092] Nucleic Acid Approaches for Antibody Delivery

[0093] The broadly neutralizing anti-HIV antibodies and antibodyfragments of the invention can also be administered to patients orsubjects as a nucleic acid preparation (e.g., DNA or RNA) that encodesthe antibody or antibody fragment, such that the patient's or subject'sown cells take up the nucleic acid and produce and secrete the encodedantibody or antibody fragment, thereby treating, inhibiting, orpreventing HIV infection.

[0094] Nucleic Acid Delivery

[0095] In the methods described above which include the administrationand uptake of exogenous DNA into the cells of a subject (i.e., genetransduction or transfection), the nucleic acids of the presentinvention can be in the form of free DNA or RNA, or the nucleic acidscan be in a vector for delivering the nucleic acids to a cell andexpressing the encoded polypeptide within a cell, whereby theantibody-encoding DNA fragment is under the transcriptional regulationof a promoter, as well as an other necessary and/or desirable componentsto regulate and/or enhance transcription and/or stability of the mRNAand to regulate and/or enhance translation of the encoded polypeptide,as would be well understood by one of ordinary skill in the art. Thevector can be a commercially available preparation, such as a plasmid oran adenovirus vector (Quantum Biotechnologies, Inc. (Laval, Quebec,Canada).

[0096] Delivery of the nucleic acid or vector to cells can be via avariety of mechanisms. As one example, delivery can be via a liposome,using commercially available liposome preparations such as LIPOFECTIN,LIPOFECTAMINE (GIBCO-BRL, Inc., Gaithersburg, Md.), SUPERFECT (Qiagen,Inc. Hilden, Germany), and TRANSFECTAM (Promega Biotec, Inc., Madison,Wis.), as well as other liposomes developed according to proceduresstandard in the art. Delivery can also be by injection (e.g., but notlimited to, intravenous or intramuscular) of naked DNA, e.g., in aplasmid or viral vector. In addition, the nucleic acid or vector of thisinvention can be delivered in vivo by electroporation, the technologyfor which is available from Genetronics, Inc. (San Diego, Calif.) aswell as by means of a SONOPORATION machine (ImaRx Pharmaceutical Corp.,Tucson, Ariz.).

[0097] As one example, vector delivery can be via a viral system, suchas a retroviral vector system which can package a recombinant retroviralgenome (see e.g., Pastan et al., Proc. Natl. Acad. Sci. U.S.A. 85:4486,1988; Miller et al., Mol. Cell. Biol. 6:2895, 1986). The recombinantretrovirus can then be used to infect and thereby deliver to theinfected cells nucleic acid encoding a broadly neutralizing antibody (oractive fragment thereof) of the invention. The exact method ofintroducing the altered nucleic acid into mammalian cells is, of course,not limited to the use of retroviral vectors. Other techniques arewidely available for this procedure including the use of adenoviralvectors (Mitani et al., Hum. Gene Ther. 5:941-948, 1994),adeno-associated viral (AAV) vectors (Goodman et al., Blood84:1492-1500, 1994), lentiviral vectors (Naidini et al., Science272:263-267, 1996), pseudotyped retroviral vectors (Agrawal et al.,Exper. Hematol. 24:738-747, 1996). Physical transduction techniques canalso be used, such as liposome delivery and receptor-mediated and otherendocytosis mechanisms (see, for example, Schwartzenberger et al., Blood87:472-478, 1996). This invention can be used in conjunction with any ofthese or other commonly used gene transfer methods.

[0098] For example, if the antibody-encoding nucleic acid of theinvention is delivered to the cells of a subject in an adenovirusvector, the dosage for administration of adenovirus to humans can rangefrom about 10⁷ to 10⁹ plaque forming units (pfu) per injection but canbe as high as 10¹² pfu per injection (Crystal, Hum. Gene Ther.8:985-1001, 1997; Alvarez and Curiel, Hum. Gene Ther. 8:597-613, 1997).A subject can receive a single injection, or, if additional injectionsare necessary, they can be repeated at six month intervals (or otherappropriate time intervals, as determined by the skilled practitioner)for an indefinite period and/or until the efficacy of the treatment hasbeen established.

[0099] Parenteral administration of the nucleic acid or vector of thepresent invention, if used, is generally characterized by injection.Injectables can be prepared in conventional forms, either as liquidsolutions or suspensions, solid forms suitable for solution ofsuspension in liquid prior to injection, or as emulsions. A morerecently revised approach for parenteral administration involves use ofa slow release or sustained release system such that a constant dosageis maintained. See, e.g., U.S. Pat. No. 3,610,795, which is incorporatedby reference herein. For additional discussion of suitable formulationsand various routes of administration of therapeutic compounds, see,e.g., Remington: The Science and Practice of Pharmacy (19th ed.) ed. A.R. Gennaro, Mack Publishing Company, Easton, Pa. 1995.

[0100] The present invention is more particularly described in thefollowing examples, which are intended as illustrative only, sincenumerous modifications and variations therein will be apparent to thoseof ordinary skill in the art.

EXAMPLE I Identification of X5, an Fab Fragment that has BroadlyNeutralizing Activity Against HIV-1

[0101] A) Materials and Methods.

[0102] Cells, viruses, plasmids, soluble CD4, gp120, gp140 and mAbs. 3T3cells expressing CD4 and CCR5 were gift from D. Littman (New YorkUniversity, NY, N.Y.). Cf2Th cells expressing high amounts of CCR5 weregift from J. Sodroski (Dana Farber Institute, Boston, Mass.); theparental cells was purchased from ATCC and used as negative control. Thestable cell line TF228 expressing LAI Env was a gift from Z. Jonak(SmithKline Beechman Pharmaceuticals, Philadelphia, Pa.) through R.Blumenthal (NCI-Frederick, Frederick, Md.). Recombinant vaccinia virusesused for the reporter gene fusion assay were described previously(Nussbaum et al. J. Virol. 68:5411-5422, 1994). Plasmids expressingvarious Envs were obtained through the NIH AIDS Research and ReferenceReagent Program or were gift from M. A. Martin (NIAID, Bethesda, Md.).Two-domain soluble CD4 (sCD4 or D1D2) (see e.g., Salzwedel et al. J.Virol. 74:326-333, 2000) was a gift from E. Berger (NIAID, Bethesda,Md.). Purified gp120_(89.6) and gp140_(89.6) were produced byrecombinant vaccinia virus (gift of R. Doms, University of Pennsylvania,Philadelphia, Pa.) with a combination of lentil lectin affinitychromatography and size exclusion chromatography. Recombinantgp120_(JRFL) was a gift from A. Schultz and N. Miller (NIAD, Bethesda,Md.). The fusion protein gp120_(Bal)-CD4 (Fouts et al. J. Virol.74:11427-11436, 2000) was a gift from T. Fouts (Institute of HumanVirology, Baltimore, Md.). The anti-CD4 polyclonal antibody T4-4 wasobtained through the AIDS Research and Reference Reagent Program from R.Sweet (SmithKline Beechman Pharmaceuticals, Philadelphia, Pa.). Theanti-gp120 mAbs 17b, 48d, 23e and 21c were gift from J. Robinson (TulaneUniversity Medical Center, New Orleans, La.). The anti-CCR5 mAb 5C7 wasa gift from L. Wu (Millenium Pharmaceuticals, Cambridge, Mass.). Thegoat polyclonal anti-CCR5 antibody CKR5(C20) was purchased from SantaCruz Biothechnology, Inc. (Santa Cruz, Calif.).

[0103] Production, purification and quantification of gp120-CD4-CCR5complexes. NIH 3T3 transfectants (10⁹ in 100 ml) expressing high amountsof CD4 and CCR5 were washed twice with cold (4° C.) phosphate-bufferedsaline (PBS) then pelleted by centrifugation and resuspended in 100 mllysis buffer (1% Brij 97, 5 mM iodoacetamide, added immediately beforeuse, 150 mM NaCl, 20 mM Tris (pH 8.2), 20 mM EDTA, and proteaseinhibitors) at 4° C. for 40 min with gentle mixing. An anti-CCR5antibody at 2 μg/ml was added to the cell suspension and incubated withgentle mixing for 4 hours at 4° C. The nuclei were pelleted bycentrifugation at 14,000 rpm for 25 min in a refrigerated centrifuge.Protein G-Sepharose beads (Sigma, St. Louis, Mo.) (1 ml) prewashed withPBS were added to the cell lysate and incubated at 4° C. for 14 hours.The beads were then washed five times with 100 ml of ice cold lysisbuffer and incubated with JRFL gp120 at 5 μg/ml in 20 ml lysis bufferfor 1 hour at 4° C. They were again washed five times with 100 coldlysis buffer, incubated with 1% formaldehyde overnight, washed twicewith cold lysis buffer and used. They contained approximately 0.01 mgCD4 and 0.02 mg gp120, as quantified by calibrating amounts of solubleCD4 and gp120. For quantification of CD4 and gp120 two duplicatedsamples each containing 0.1% of the total amount of bead-associatedgp120-CD4-CCR5 complexes were used. They were eluted by adding 4× samplebuffer for SDS-PAGE gel and kept overnight at 37° C. They were run on a10% SDS-PAGE gel simultaneously with calibrating amounts (1, 3, 10, 30,100 ng) of soluble four-domain CD4 (sCD4) (see, e.g., Deen et al. Nature331:82-84, 1988) or gp120 and were electrophoretically transferred tonitrocellulose membranes. The membranes were blocked with 20 mM tris-HCl(pH 7.6) buffer containing 140 mM NaCl, 0.1% Tween-20 and 5% nonfatpowdered milk. For Western blotting these membranes were incubated withanti-CD4 or anti-gp120 antibodies, then washed and incubated withhorseradish peroxidase (HRP)-conjugated secondary antibodies. They weredeveloped by using the supersignal chemiluminescent substrate fromPierce (Rockford, Ill.). The images were acquired by a BioRadphosphoimager (BioRad, Hercules, Calif.). The signal from thecalibrating molecules was integrated for each band and plotted in acalibration curve for the signal vs. amount dependence. The amounts ofCD4 and gp120 were then calculated by interpolation using thecalibration curve.

[0104] Screening of the phage display library. A phage library (IgG1k)from a seropositive individual with a relatively high cross-cladeneutralizing antibody titer (FDA2), constructed as described (Burton etal. Proc. Natl. Acad. Sci. USA 88:10134-10137, 1991; and C. F. Barbas,D. R. Burton, J. K. Scott, G. J. Silverman, Phage Display: A LaboratoryManual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,2001) was used. Phages (50 μl) were preadsorbed on protein G beads inPBS for 1 h at 37° C. Unbound phages were recovered by centrifugation(1500 rpm for 5 min at 4° C.) and then incubated with protein G beadsassociated with gp120-CD4-CCR5 complexes for 2 hours at 4° C. undergentle agitation. Beads were washed 10 times with PBS containing 0.5%Tween.

[0105] Phages were eluted from the beads by incubation with 50 μl 0.1MHCl-glycine (pH 2.2) solution containing BSA at 1 mg/ml for 10 min atroom temperature. The solution was neutralized with 3 μl of 2MTRIS-base. XL1-Blue E. coli cells were reinfected and panning repeatedfor total of 5 rounds of panning.

[0106] Preparation of soluble Fab fragments. Phagemid DNA was isolatedand digested with Spe I and Nhe I to remove the gene III fragment andself-religated as described elsewhere (Barbas et al. Proc. Natl. Acad.Sci. US.A. 88:7978-7982, 1991). The Δgene III-phagemid library was usedto transform XL1-Blue E. coli cells to produce clones secreting Fabfragments. 60 such clones were grown up and the corresponding Fabs wereobtained by lysing the cell pellet. Cells were frozen in a dryice-ethanol bath for 5 min followed by thawing in a 37° C. water bath.This process was repeated four times and the cell debris were pelletedby centrifugation at 15,000 rpm for 15 minutes at 4° C.

[0107] ELISA analysis of Fab supernatants. ELISA wells were coatedovernight at 4° C. with 50 μl of gp120 (10 μg/ml in PBS), blocked in 100μl of 3% BSA/PBS for 1 hour at RT. After 5 washes with 0.05% Tween20/PBS(Washing Buffer, WB), wells were incubated with 50 μl Fab supernatantsfor 1 hour at RT. After 10 washes with WB, 50 μl of a 1:1000 dilution ofalkaline phosphatase-conjugated goat anti-human IgG F(ab′)₂ was addedand incubated for 1 hour at RT. Following 10 washes with WB, the assaywas developed with p-nitrophenyl phosphate substrate (Sigma, St. Louis,Mo.) and monitored at 405 nm. Heavy chains from positive clones weresequenced using the SeqGz primer (5′-GAAGTAGTCCTTGACCAG-3′; SEQ ID NO:1).

[0108] Production and purification of Fab. The selected phage wasamplified and purified by standard methods (see C. F. Barbas, D. R.Burton, J. K. Scott, G. J. Silverman, Phage Display: A LaboratoryManual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,2001). Protein G columns were used for purification.

[0109] Production and purification of detergent solubilized CCR5(dsCCR5). DsCCR5 was produced by a methodology adapted from thatdescribed previously (Mirzabekov et al. J. Biol. Chem. 274:28745-28750,1999).

[0110] Cell-cell fusion inhibition assay. The gene reporter cell-cellfusion assay was previously described (Nussbaum et al. J. Virol.68:5411-5422, 1994). Briefly, recombinant vaccinia viruses atmultiplicity of infection 10 were used to infect the target (vCB21R) andeffector cells (vTF 7.3 plus virus expressing the HIV-1 Env). The β-galfusion assay was performed two hours after mixing the cells. The extentof fusion was quantitated calorimetrically. Fusion induced by sCD4 wasperformed by incubation of 10⁵ effector cells expressing the Env withsCD4 (1 ug/ml) at 37° C. for 30 min before mixing with the target cells(Salzwedel et al. J. Virol. 74:326-333, 2000). The inhibitory effect ofX5 was evaluated by mixing the effector cells with X5 for 30 min at 37°C. and then performing the fusion assay.

[0111] HIV neutralization assay. Virus neutralization assays wereperformed by using infection with a luciferase reporter HIV-1 Envpseudotyping system (Connor et al. Virology 206:935-944, 1995). Viralstocks were prepared by transfecting 293T cells with plasmids encodingthe luciferase virus backbone (pNL-Luc-ER) and Env from various HIVstrains. The resulting supernatant was clarified by centrifugation for10 min at 2,000 rpm in a Sorvall RT-7 centrifuge (RTH-750 rotor), passedthrough a 45-um pore size sterile filter (Millipore, Bedford, Mass.) andused or stored at −80° C. The virus was pre-incubated with variousconcentrations of X5 (0.1, 1.0, 10.0, 50, and 80 or 100 ug/ml) for 30min at 37° C. Cells were then infected with 50 μl of virus preparationfor 4 h at 37° C., then fresh media was added and incubation wascontinued for 48 h. Cells were then washed with PBS and lysed withluciferase assay buffer (Promega, Madison, Wis.). Luciferase activitywas determined by adding 50 μl of freshly prepared luciferase assaysubstrate to 50 μl of cell lysate and measuring the intensity ofchemiluminescence in a LumiCount microplate luminometer (Turner Designs,Sunnyvale, Calif.). All experiments were performed at least intriplicate and the results expressed as relative light units (RLU) persecond.

[0112] ELISA binding assay. The assay used for binding is a modifiedELISA type assay. Briefly, gp120 or sCD4-gp120 was non-specificallyattached to the bottom of 96-well plates by incubation of 0.1 mlsolution containing 100 ng of the protein at 4° C. overnight. Plateswere then treated with 4% non-fat milk (Biorad) in order to preventnonspecific binding. The plates were washed with TBS containing 0.1%Tween-20; dsCCR5 in Cymal lysis buffer (1% Cymal, 100 mM (NH₄)₂SO₄, 20mM Tris, 10% glycerol) was then added and incubated at 4° C. overnight.Unbound molecules of dsCCR5 were washed and X5 was added. After washingbound X5 was detected by anti-human IgG.

[0113] The antibody competition experiment was performed by using amixture of the competing Ab (at different concentrations) with 0.5 ug/mlbiotinylated X5 instead of X5 alone following the procedure as describedabove. Biotinylated proteins were prepared by incubation with 2 mMbiotin (prepared from solid NHS-LS-Biotin (Pierce, Calif.) dissolved at200 mM in DMSO as stock solution) on wet ice for 1 h. The biotinylationwas quenched with 20 mM glycine on ice for 15 min. Binding ofbiotinylated X5 was detected using streptavidin-HRP secondary antibody.

[0114] Flow cytometry. Cells (typically 0.5×10⁶) were incubated for 1 hon ice with the antigen specific antibodies. They were washed, andincubated for another hour on ice with rabbit IgG (10 μg/ml) (Sigma, St.Louis, Mo.) to improve the specificity, then washed and incubated for 1h with an anti-mouse phycoerythrin-conjugated polyclonal antibody(Sigma). The cells were washed and fixed with paraformaldehyde on icefor 10 min. The flow cytometry measurements were performed withFACSCalibur (Becton Dickinson, San Jose, Calif.).

[0115] Binding of Env-specific antibodies to HIV-1-infected cells. TheT-cell line H9 (a gift from Q.Sattentau and supplied by the MRC AIDSReagent Project) was grown in RPMI 1640 supplemented with 10% fetal calfserum. H9 cells were infected with the HIV-1 TCLA X4 MN isolate(obtained through the AIDS Research and Reference Reagent Program fromR. Gallo) for 10 days to achieve high level of Env expression asdetected by gp120-specific mAbs and flow cytometry (see above). Then theantibodies were added at various concentrations, the cells were washedtwice, and the bound human antibodies detected using anti-human IgG byflow cytometry as described above.

[0116] B) Results.

[0117] Selection of a phage (X5) with high affinity to gp120-CD4-CCR5complexes. For panning we used complexes containing about 0.01 mg CD4and 0.02 mg JRFL gp120. The amount of CCR5 was not determined preciselybut was about 0.01 mg as found by comparison of CCR5 Western blots ofknown amounts of detergent-solubilized CCR5. After 5 rounds of panningone phage was selected. This phage, denoted X5, was amplified. X5exhibited binding activity to protein G cross-linked to Sepharose beadswith an affinity (equilibrium dissociation constant) of 15 nM.Phage-displayed X5 had 15-fold lower effective affinity (1.4 nM) forprotein G beads than for the gp120-CD4-CCR5 complex (0.09 nm). The X5Fab was purified by using protein G columns.

[0118] Binding of X5 to a CD4-inducible epitope on gp120 that isenhanced by dsCCR5. To find whether the Fab of the selected phage wasable to bind gp120 and its complexes with CD4 and CCR5 we used anELISA-type of assay. X5 bound gp120 from several isolates and itsbinding was increased 5-10-fold after binding of sCD4 (D1D2) to gp120(FIGS. 1 and 2). The affinity of binding (equilibrium dissociationconstants) to gp120_(89.6) and sCD4-gp120_(89.6) complexes was 9.4 nMand 1 nM (FIG. 2), and for JRFL-10 and 2 nM, respectively (FIG. 1).Binding of dsCCR5 further enhanced the X5 epitope exposure by 30-60%(FIG. 1). Similar affinities were observed for binding to oligomeric,fusion-active gp120-gp41_(MN) expressed at the surface of chronicallyinfected H9 cells (FIG. 3). For this experimental system the X5 affinityin presence of sCD4 was comparable to that of CD4bs-specific mAb IgG1b12in the absence of sCD4 and several fold higher than the affinity of 17bwhich was previously reported to exhibit an increased affinity to thegp120-CD4 complex (Thali et al. J. Virol. 67:3978-3988, 1993). Theaffinity of X5 for the gp120_(JRFL)-sCD4-CCR5 complex used for the X5selection was 1 nM and the effective affinity of phage-displayed X5 forthe gp120_(JRFL)-sCD4-CCR5 complex was 0.09 nM. Thus X5 exhibit thehighest affinity to the gp120-sCD4 complex among known antibodies toCD4-inducible epitopes. The affinity was not significantly dependent onthe tropism of the Envs.

[0119] Recombinant gp120 and gp140 from several primary isolates (a giftfrom C. Broder) behaved similarly. In all (92UG037.8 (Clade A, R5);92HT593.1 (Clade B, R5 X4); 93ZR001.3 (Clade D, R5 X4) and 89.6) casesbinding to gp120 was higher than binding to gp140 suggesting effects ofgp41 or/and oligomerization. Therefore, the X5 epitope is a conservedconformational epitope that is induced by CD4 and enhanced by CCR5. Thisis the first example of an epitope for which exposure is enhanced byCCR5. These results suggest the existence of a very early intermediatein the HIV-1 entry process that is induced by receptor molecules.

[0120] Inhibition of HIV-1 envelope glycoprotein-mediated cell fusionand entry. To find whether X5 inhibits HIV-1 Env-mediated fusion we usedseveral assays. In a sCD4-induced fusion assay X5 inhibited almostcompletely sCD4-mediated fusion mediated by the dual tropic primaryisolate Env 89.6 at very low (100 ng/ml) concentrations (FIG. 4).Similar results were obtained for several X4, R5 and X4R4 Envs (Table1). Inhibition of fusion mediated by X4 Envs was somewhat less efficientcompared to R5 Env-mediated fusion. Fusion mediated by cell-associatedCD4 required on average 10-fold higher concentrations of X5. This may berelated to the fact that cell-cell fusion is difficult to inhibitespecially when the surface concentrations of CD4 and CCR5 (CXCR4) arehigh. X5 was able to neutralize several representative R5 (JRFL andBal), X4 (NL4-3) and X4R5 (89.6) isolates of HIV-1 at concentrations inthe range of 1 to 10 μg/ml (FIG. 5). These results indicate that X5 is anovel broadly neutralizing HIV-1 antibody which can be used as anefficient inhibitor of HIV-1 infections.

[0121] Table 1: Inhibition of sCD4-induced R5, X4, and R5-X4Envs-mediated fusion by X5. The data are represented as % of controlwithout X5; “-” denotes no fusion in the control. 293T cells weretransfected by plasmids encoding various Envs. They were pre-incubatedwith sCD4 (1 ug/ml) and X5 (125 ng/ml) for 30 min at 37° C. andincubated with the target cells (SupT1 cells expressing CXCR4 and NIH3T3 CD4.CCR5 cells) for 2 h at 37° C. Fusion was measured by thereporter gene fusion assay. Envs NL4-3 HXB2 89.6 JRFL ADA Bal SF162 R5 —— 99 98 96 96 100 X4 83 71 79 — — — —

[0122] Characterization of the X5 epitope. To characterize the X5binding site on gp120, we used several human monoclonal antibodies (giftof J. Robinson) against CD4-inducible epitopes. Two of these antibodiesdid not compete with X5 in the ELISA assay. Very low competition wasobserved with 17b at high concentrations (FIGS. 6A and 6B), suggestingslight epitope overlap or steric interference. Although we were not ableto localize precisely the X5 epitope it appears that it is close to thegp41 association site with gp120, as indicated by the poorer binding ofall gp140 Envs compared to gp120s. Studies of X5 competition with moreantibodies with known epitopes and the X5 crystal structure incombination with the known gp120 core structure will facilitatedetermination of the precise epitope recognized by X5. The amino acidsequences of the X5 light and heavy chains are shown in FIGS. 7 and 8,respectively.

EXAMPLE II Generation of X5 Antibody Sequence Variants with IncreasedAffinity for Env-CD4-Co-Receptor Complexes

[0123] The amino acid sequence of any antibody or antibody fragment ofthe invention may be varied in order to generate variant antibodies withequivalent or improved affinity for Env-CD4-co-receptor complexes. Suchvariant antibodies can be created and tested for their relative affinityusing well-known methods and/or methods described herein. Suchtechniques are described, e.g., in Daugherty et al. (“Quantitativeanalysis of the effect of the mutation frequency on the affinitymaturation of single chain Fv antibodies.” Proc. Natl. Acad. Sci. USA97(5):2029-34, 2000); Cherry et al. (“Directed evolution of a fungalperoxidase.” Nat. Biotechnol. 17(4): 379-84, 1999); and Vartanian et al.(“Hypermutagenic PCR involving all four transitions and a sizeableproportion of transversions.” Nucleic Acids Res. 24(14):2627-31, 1996).

[0124] Random mutagenesis of the X5 antibody gene was used to identifyX5 sequence variants with improved affinity for Env-CD4-CCR5 and Env-CD4complexes. Two phage-X5 mutagenesis libraries, one expressing antibodyX5 sequence variants as Fab fragments and the other expressing antibodyX5 as scFv (single chain antibody) fragments, were constructed andpanned sequentially against both complexes using methods similar tothose described in Example I above. One Fab clone was selected from theX5-Fab mutagenesis library. The new X5-Fab clone, designated FabS,showed five-fold increased affinity for Env-CD4-CCR5 complex, comparedto the original X5-Fab. The mutations were located in the joint regionbetween the heavy chain variable region and the first constant domain(CH1), in which the alanine in the original X5 sequence was changed toproline and the serine in the original sequence was changed to glycine(SEQ ID NO: 11; FabS heavy chain sequence), as shown in FIG. 4. Thenucleotide sequence encoding the light and heavy chains of FabS is setforth in SEQ ID NO: 12.

EXAMPLE III Generation of Antibody Fusion Polypeptides Based on the X5Antibody Sequence

[0125] Fusion proteins comprising antibody fragments and otherfunctional domains which increase the efficacy of the antibody intreating, inhibiting, or preventing HIV infection are contemplated bythe present invention. Such antibodies fusion proteins can be made usingstandard techniques that are well known in the art, and used in themethods of the invention for the treatment of and prophylaxis againstinfection by HIV. Below are several examples of fusion proteins basedupon the X5 antibody fragment described in Example I above. Two basictypes of fusion proteins are shown: those based upon single-chainantibody (ScFv) fragments, and those based upon Fab fragments. One ofordinary skill in the art will understand that any antibody or antibodyfragment of the invention can be used to generate these and other typesof fusion proteins.

[0126] The first example of such a fusion protein is a ScFvX5-CD4 fusionprotein, i.e., a single-chain antibody (ScFv) fragment comprising adomain from the variable region of the X5 heavy chain (VH) and a domainfrom the variable region of the X5 light chain (VL), fused to fused tosoluble CD4 (sCD4). The HV and LV domains are separated by a fifteenamino acid long flexible linker consisting of three repeats of thepentapeptide Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 21), and the VL and sCD4domains of the fusion protein are separated by a variable lengthflexible linker (e.g., containing 20, 30, or 40 amino acid residues)containing, e.g., four, six, or eight repeats of Gly-Gly-Gly-Gly-Ser.The components of this fusion protein, labeled a through e, are asfollows. Although the example below shows the VH domain as beingpositioned amino-terminal to the VL domain (VH-Linker (L3)-VL-), one ofordinary skill in the art will readily recognize that the relativepositions of the VH and VL domains can be swapped (e.g., VL-Linker(L3)-VH-) for all of the exemplary fusion protein constructs shownbelow.

[0127] A) ScFvX5-CD4 Fusion Protein

[0128] Construct: VH-Linker (L3)-VL-Linker(variable length)-sCD4(a-b-c-d-e)

[0129] a) Variable Heavy Chain (VH) (SEQ ID NO: 14) M A V Q L L E Q S GA E V K K P G S S V Q V S C K A S G G T F S M Y G F N W V R Q A P G H GL E W M G G I I P I F G T S N Y A Q K F R G R V T F T A D Q A T S T A YM E L T N L R S D D T A V Y Y C A R D F G P D W E D G D S Y D G S G R GF F D F W G Q G T L V T V S S

[0130] b) Linker (L) (SEQ ID NO: 21)

[0131] (Gly₄Ser)×3

[0132] c) Variable Light Chain (VL) (SEQ ID NO: 15) D I V L T Q S P G TL S L S A G E R A T L S C R A S Q S V S S G S L A W Y Q Q K P G Q A P RL L I Y G A S T R A T G I P D R F S G S G S G T D F T L T I G R L E P ED L A V Y Y C Q Q Y G T S P Y T F G Q G T K L E I K R T

[0133] d) Linker (Variable length—20 or 30 or 40 a.a Long) (SEQ ID NO:16, 17, or 18)

[0134] (Gly₄Ser)×4 or 6 or 8

[0135] e) sCD4 (Two Domain Soluble CD4) (SEQ ID NO: 19) M N R G V P F RH L L L V L Q L A L L P A A T Q G K K V V L G K K G D T V E L T C T A SQ K K S I Q F H W K N S N Q I K I L G N Q G S F L T K G P S K L N D R AD S R R S L W D Q G N F P L I I K N L K I E D S D T Y I C E V E D Q K EE V Q L L V F G L T A N S D T H L L Q G Q S L T L T L E S P P G S S P SV Q C R S P R G K N I Q G G K T L S V S Q L E L Q D S G T W T C T V L QN Q K K V E F K I D I V V L

[0136] Below is a second example of a fusion polypeptide that, inaddition to the above components, also contains a synthetic peptide,T20, which corresponds to a peptide sequence found in HIV-1 gp41, and isa strong inhibitor of HIV-1 viral fusion (see, e.g., Lawless et al.Biochemistry 35:13697-13708, 1996).

[0137] B) ScFv-CD4-T20 Fusion Protein

[0138] Construct: VH-Linker (L3)-VL-Linker(variable length)-sCD4-L1-T20(a-b-c-d-e-f-g)

[0139] This construct is identical to construct A above, but inaddition, after sCD4, contains another linker and the T-20 peptide:

[0140] f) Linker (SEQ ID NO: 13)

[0141] (Gly₄Ser)×1

[0142] g) T-20Peptide (SEQ ID NO: 20) Y T S L I H S L I E E S Q N Q Q EK N E Q E L L E L D K W A S L W N W F

[0143] Below is an example of a fusion polypeptide containing an X5 Fabfragment, i.e., the X5 light chain fragment (VLCL) containing thevariable region and the constant region, which is disulfide-bonded(represented below by “s-s”) to the heavy chain fragment containing thevariable region and the CH1 portion of the constant region (VHCH1). Oneof skill in the art will recognize that X5 Fabs can contain heavy andlight chains containing PelB, OmpA, or other signal sequences thatdirect protein secretion, e.g., as set forth in SEQ ID NOs: 3 and 2,respectively, or can contain heavy and light chains that lack suchsignal sequences, e.g., as set forth in SEQ ID NOs:14 and 15,respectively.

[0144] The X5 Fab fusion protein shown below contains a soluble CD4domain fused to the carboxy terminus of the X5 heavy chain. Constructscontaining the sCD4 domain fused to the X5 light chain instead of theheavy chain may also be used.

[0145] C) Fab′-CD4 Fusion Protein

[0146] Construct: VLCL-s-s-VHCH1-Linker(variable length)-sCD4

[0147] a) X5 Fab Sequence (e.g., SEQ ID NOs: 3 Plus 2 or SEQ ID NOs: 14Plus 15).

[0148] b) Linker (Variable length—20 or 30 or 40 a.a Long) (SEQ ID NO:16, 17, or 18)

[0149] (Gly₄Ser)×4 or 6 or 8

[0150] c) sCD4 (Two Domain) (SEQ ID NO: 19) M N R G V P F R H L L L V LQ L A L L P A A T Q G K K V V L G K K G D T V E L T C T A S Q K K S I QF H W K N S N Q I K I L G N Q G S F L T K G P S K L N D R A D S R R S LW D Q G N F P L I I K N L K I E D S D T Y I C E V E D Q K E E V Q L L VF G L T A N S D T H L L Q G Q S L T L T L E S P P G S S P S V Q C R S PR G K N I Q G G K T L S V S Q L E L Q D S G T W T C T V L Q N Q K K V EF K I D I V V L

[0151] The fourth construct is the same as the X5 Fab construct above,except it also contains a T20 domain. The T20 domain, like the sCD4domain, may also be fused to either the heavy chain or the light chain.The construct below shows the T20 domain directly connected to the sCD4domain by a flexible linker; however, constructs with the sCD4 domain onone chain and the T20 domain on the other chain (connected to theremainder of the chain by a flexible linker) are also included in thepresent invention.

[0152] D) Fab′-CD4-T20 Fusion Protein

[0153] Construct: VLCL-s-s-VHCH1-Linker(variable length)-sCD4-L1-T20

[0154] This construct is the same as construct C above, but, inaddition, after sCD4 contains:

[0155] d) Linker (SEQ ID NO: 13)

[0156] (Gly₄Ser)×1

[0157] e) T-20Peptide (SEQ ID NO: 20)

[0158] YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF

SEQUENCES

[0159] Amino Acid Sequence of X5 Light Chain Including OmpA Signal (SEQID NO: 2) M K K T A I A I A V A L A G F A T V A Q A A E L V L T Q S P GT L S L S A G E R A T L S C R A S Q S V S S G S L A W Y Q Q K P G Q A PR L L I Y G A S T R A T G I P D R F S G S G S G T D F T L T I G R L E PE D L A V Y Y C Q Q Y G T S P Y T F G Q G T K L E I K R T V A A P S V FI F P P S D E Q L K S G T A S V V C L L N N F Y P R E A K V Q W K V D NA L Q S G N S Q E S V T E H D S R D S T Y S L G S T L T L S K A D Y E KH K V Y A C E V T H Q G L S S P V T K S F N R G E C

[0160] Amino Acid Sequence of X5 Heavy Chain, Including PelB Signal (SEQID NO: 3) M K Y L L P T A A A G L L L L A A Q P A M A E V Q L L E Q S GA E V K K P G S S V Q V S C K A S G G T F S M Y G F N W V R Q A P G H GL E W M G G I I P I F G T S N Y A Q K F R G R V T F T A D Q A T S T A YM E L T N L R S D D T A V Y Y C A R D F G P D W E D G D S Y D G S G R GF F D F W G Q G T L V T V S S A S T K G P S V F P L A P S S K S T S G GT A A L G C L V K D Y F P E P V T V S W N S G A L T S G V H T F P A V LQ S S G L Y S L S S V V T V P S S S L G T Q T Y I C N V N H K P S N T KV D K K V E P K S C D K T S

[0161] Nucleotide Sequence of X5 (SEQ ID NO: 4)

[0162] Length: 1539 1 atgaaaaaga cagctatcgc gattgcagtg gcactggctggtttcgctac 51 cgtggcccag gcggccgagc tcgtgttgac acagtctcca ggcaccctgt 101ctttgtctgc aggggaaaga gccaccctct cctgcagggc cagtcagagt 151 gttagcagcggctccttagc ctggtaccag cagaaacctg gtcaggctcc 201 caggctcctc atctacggtgcatccaccag ggccactggc atcccagaca 251 ggttcagtgg cagtgggtct gggacagacttcactctcac aatcggcaga 301 ctggagcctg aagatctcgc agtatattac tgtcagcagtatggtacctc 351 accgtacact tttggccagg ggaccaaact ggagatcaaa cgaactgtgg401 ctgcaccatc tgtcttcatc ttcccgccat ctgatgagca gttgaaatct 451ggaactgcct ctgttgtgtg cctgctgaat aacttctatc ccagagaggc 501 caaagtacagtggaaggtgg ataacgccct ccaatcgggt aactcccagg 551 agagcgtcac agagcatgacagcagggaca gcacctacag cctcggcagc 601 accctgacgc tgagcaaagc agactacgagaaacacaaag tctacgcctg 651 cgaagtcacc catcagggcc tgagttcgcc cgtcacaaagagcttcaaca 701 ggggagagtg ttaattctag ataattaatt aggaggaatt taaaatgaaa751 tacctattgc ctacggcagc cgctggattg ttattactcg ctgcccaacc 801agccatggcc gaggtgcagc tgctcgagca gtctggggct gaggtgaaga 851 agcctgggtcctcggtgcag gtctcctgca aggcctctgg aggcaccttc 901 agcatgtatg gtttcaactgggtgcgacag gcccctggac atggccttga 951 gtggatggga gggatcatcc ctatctttggtacatcaaac tacgcacaga 1001 agttccgggg cagagtcacg tttaccgcgg accaagccacgagcacagcc 1051 tacatggagc tgaccaacct gcgatctgac gacacggccg tctattattg1101 tgcgagagat tttggccccg actgggaaga cggtgattcc tatgatggta 1151gtggccgggg gttctttgac ttctggggcc agggaaccct ggtcaccgtc 1201 tcctctgcctccaccaaggg cccatcggtc ttccccctgg caccctcctc 1251 caagagcacc tctgggggcacagcggccct gggctgcctg gtcaaggact 1301 acttccccga accggtgacg gtgtcgtggaactcaggcgc cctgaccagc 1351 ggcgtgcaca ccttcccggc tgtcctacag tcctcaggactctactccct 1401 cagcagcgtg gtgaccgtgc cctccagcag cttgggcacc cagacctaca1451 tctgcaacgt gaatcacaag cccagcaaca ccaaggtgga caagaaagtt 1501gagcccaaat cttgtgacaa aactagctaa ttaatttaa

[0163] Amino Acid Sequence of CDR3 Region of X5 Heavy Chain (SEQ ID NO:5) D F G P D W E D G D S Y D G S G R G F F D F

[0164] Amino Acid Sequence of CDR2 Region of X5 Heavy Chain (SEQ ID NO:6) G I I P I F G T S N Y A Q K F R G

[0165] Amino Acid Sequence of CDR1 Region of X5 Heavy Chain (SEQ ID NO:7)

[0166] M Y G F N

[0167] Amino Acid Sequence of CDR3 Region of X5 Light Chain (SEQ ID NO:8) Q Q Y G T S P Y T F G Q G T K L E I K R

[0168] Amino Acid Sequence of CDR2 Region of X5 Light Chain (SEQ ID NO:9) G A S T R A T G I

[0169] Amino Acid Sequence of CDR1 Region of X5 Light Chain (SEQ ID NO:10) R A S Q S V S S G S L A W

[0170] Amino Acid Sequence of FabS Heavy Chain, Including PelB Signal(SEQ ID NO: 11) M K Y L L P T A A A G L L L L A A Q P A M A E V Q L L EQ S G A E V K K P G S S V Q V S C K A S G G T F S M Y G F N W V R Q A PG H G L E W M G G I I P I F G T S N Y A Q K F R G R V T F T A D Q A T ST A Y M E L T N L R S D D T A V Y Y C A R D F G P D W E D G D S Y D G SG R G F F D F W G Q G T L V T V S S P G T K G P S V F P L A P S S K S TS G G T A A L G C L V K D Y F P E P V T V S W N S G A L T S G V H T F PA V L Q S S G L Y S L S S V V T V P S S S L G T Q T Y I C N V N H K P SN T K V D K K V E P K S C D K T S

[0171] Nucleotide Sequence of FabS (SEQ ID NO: 12) 1 atgaaaaagacagctatcgc gattgcagtg gcactggctg gtttcgctac 51 cgtggcccag gcggccgagctcgtgttgac acagtctcca ggcaccctgt 101 ctttgtctgc aggggaaaga gccaccctctcctgcagggc cagtcagagt 151 gttagcagcg gctccttagc ctggtaccag cagaaacctggtcaggctcc 201 caggctcctc atctacggtg catccaccag ggccactggc atcccagaca251 ggttcagtgg cagtgggtct gggacagact tcactctcac aatcggcaga 301ctggagcctg aagatctcgc agtatattac tgtcagcagt atggtacctc 351 accgtacacttttggccagg ggaccaaact ggagatcaaa cgaactgtgg 401 ctgcaccatc tgtcttcatcttcccgccat ctgatgagca gttgaaatct 451 ggaactgcct ctgttgtgtg cctgctgaataacttctatc ccagagaggc 501 caaagtacag tggaaggtgg ataacgccct ccaatcgggtaactcccagg 551 agagcgtcac agagcatgac agcagggaca gcacctacag cctcggcagc601 accctgacgc tgagcaaagc agactacgag aaacacaaag tctacgcctg 651cgaagtcacc catcagggcc tgagttcgcc cgtcacaaag agcttcaaca 701 ggggagagtgttaattctag ataattaatt aggaggaatt taaaatgaaa 751 tacctattgc ctacggcagccgctggattg ttattactcg ctgcccaacc 801 agccatggcc gaggtgcagc tgctcgagcagtctggggct gaggtgaaga 851 agcctgggtc ctcggtgcag gtctcctgca aggcctctggaggcaccttc 901 agcatgtatg gtttcaactg ggtgcgacag gcccctggac atggccttga951 gtggatggga gggatcatcc ctatctttgg tacatcaaac tacgcacaga 1001agttccgggg cagagtcacg tttaccgcgg accaagccac gagcacagcc 1051 tacatggagctgaccaacct gcgatctgac gacacggccg tctattattg 1101 tgcgagagat tttggccccgactgggaaga cggtgattcc tatgatggta 1151 gtggccgggg gttctttgac ttctggggccagggaaccct ggtcaccgtc 1201 tcctctcccg ggaccaaggg cccatcggtc ttccccctggcaccctcctc 1251 caagagcacc tctgggggca cagcggccct gggctgcctg gtcaaggact1301 acttccccga accggtgacg gtgtcgtgga actcaggcgc cctgaccagc 1351ggcgtgcaca ccttcccggc tgtcctacag tcctcaggac tctactccct 1401 cagcagcgtggtgaccgtgc cctccagcag cttgggcacc cagacctaca 1451 tctgcaacgt gaatcacaagcccagcaaca ccaaggtgga caagaaagtt 1501 gagcccaaat cttgtgacaa aactagctaattaatttaa

[0172] Incorporation By Reference

[0173] All publications and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each independent publication or patent application was specificallyand individually indicated to be incorporated by reference.

[0174] Other Embodiments

[0175] While the invention has been described in connection withspecific embodiments thereof, it will be understood that it is capableof further modifications and this application is intended to cover anyvariations, uses, or adaptations of the invention following, in general,the principles of the invention and including such departures from thepresent disclosure come within known or customary practice within theart to which the invention pertains and may be applied to the essentialfeatures hereinbefore set forth, and follows in the scope of theappended claims.

1 21 1 18 DNA Artificial Sequence Synthetic 1 gaagtagtcc ttgaccag 18 2237 PRT Artificial Sequence Synthetic 2 Met Lys Lys Thr Ala Ile Ala IleAla Val Ala Leu Ala Gly Phe Ala 1 5 10 15 Thr Val Ala Gln Ala Ala GluLeu Val Leu Thr Gln Ser Pro Gly Thr 20 25 30 Leu Ser Leu Ser Ala Gly GluArg Ala Thr Leu Ser Cys Arg Ala Ser 35 40 45 Gln Ser Val Ser Ser Gly SerLeu Ala Trp Tyr Gln Gln Lys Pro Gly 50 55 60 Gln Ala Pro Arg Leu Leu IleTyr Gly Ala Ser Thr Arg Ala Thr Gly 65 70 75 80 Ile Pro Asp Arg Phe SerGly Ser Gly Ser Gly Thr Asp Phe Thr Leu 85 90 95 Thr Ile Gly Arg Leu GluPro Glu Asp Leu Ala Val Tyr Tyr Cys Gln 100 105 110 Gln Tyr Gly Thr SerPro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu 115 120 125 Ile Lys Arg ThrVal Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser 130 135 140 Asp Glu GlnLeu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn 145 150 155 160 AsnPhe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala 165 170 175Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu His Asp Ser Arg 180 185190 Asp Ser Thr Tyr Ser Leu Gly Ser Thr Leu Thr Leu Ser Lys Ala Asp 195200 205 Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu210 215 220 Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230235 3 261 PRT Artificial Sequence Synthetic 3 Met Lys Tyr Leu Leu ProThr Ala Ala Ala Gly Leu Leu Leu Leu Ala 1 5 10 15 Ala Gln Pro Ala MetAla Glu Val Gln Leu Leu Glu Gln Ser Gly Ala 20 25 30 Glu Val Lys Lys ProGly Ser Ser Val Gln Val Ser Cys Lys Ala Ser 35 40 45 Gly Gly Thr Phe SerMet Tyr Gly Phe Asn Trp Val Arg Gln Ala Pro 50 55 60 Gly His Gly Leu GluTrp Met Gly Gly Ile Ile Pro Ile Phe Gly Thr 65 70 75 80 Ser Asn Tyr AlaGln Lys Phe Arg Gly Arg Val Thr Phe Thr Ala Asp 85 90 95 Gln Ala Thr SerThr Ala Tyr Met Glu Leu Thr Asn Leu Arg Ser Asp 100 105 110 Asp Thr AlaVal Tyr Tyr Cys Ala Arg Asp Phe Gly Pro Asp Trp Glu 115 120 125 Asp GlyAsp Ser Tyr Asp Gly Ser Gly Arg Gly Phe Phe Asp Phe Trp 130 135 140 GlyGln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 145 150 155160 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 165170 175 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr180 185 190 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr PhePro 195 200 205 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser ValVal Thr 210 215 220 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile CysAsn Val Asn 225 230 235 240 His Lys Pro Ser Asn Thr Lys Val Asp Lys LysVal Glu Pro Lys Ser 245 250 255 Cys Asp Lys Thr Ser 260 4 1539 DNAArtificial Sequence Synthetic 4 atgaaaaaga cagctatcgc gattgcagtggcactggctg gtttcgctac cgtggcccag 60 gcggccgagc tcgtgttgac acagtctccaggcaccctgt ctttgtctgc aggggaaaga 120 gccaccctct cctgcagggc cagtcagagtgttagcagcg gctccttagc ctggtaccag 180 cagaaacctg gtcaggctcc caggctcctcatctacggtg catccaccag ggccactggc 240 atcccagaca ggttcagtgg cagtgggtctgggacagact tcactctcac aatcggcaga 300 ctggagcctg aagatctcgc agtatattactgtcagcagt atggtacctc accgtacact 360 tttggccagg ggaccaaact ggagatcaaacgaactgtgg ctgcaccatc tgtcttcatc 420 ttcccgccat ctgatgagca gttgaaatctggaactgcct ctgttgtgtg cctgctgaat 480 aacttctatc ccagagaggc caaagtacagtggaaggtgg ataacgccct ccaatcgggt 540 aactcccagg agagcgtcac agagcatgacagcagggaca gcacctacag cctcggcagc 600 accctgacgc tgagcaaagc agactacgagaaacacaaag tctacgcctg cgaagtcacc 660 catcagggcc tgagttcgcc cgtcacaaagagcttcaaca ggggagagtg ttaattctag 720 ataattaatt aggaggaatt taaaatgaaatacctattgc ctacggcagc cgctggattg 780 ttattactcg ctgcccaacc agccatggccgaggtgcagc tgctcgagca gtctggggct 840 gaggtgaaga agcctgggtc ctcggtgcaggtctcctgca aggcctctgg aggcaccttc 900 agcatgtatg gtttcaactg ggtgcgacaggcccctggac atggccttga gtggatggga 960 gggatcatcc ctatctttgg tacatcaaactacgcacaga agttccgggg cagagtcacg 1020 tttaccgcgg accaagccac gagcacagcctacatggagc tgaccaacct gcgatctgac 1080 gacacggccg tctattattg tgcgagagattttggccccg actgggaaga cggtgattcc 1140 tatgatggta gtggccgggg gttctttgacttctggggcc agggaaccct ggtcaccgtc 1200 tcctctgcct ccaccaaggg cccatcggtcttccccctgg caccctcctc caagagcacc 1260 tctgggggca cagcggccct gggctgcctggtcaaggact acttccccga accggtgacg 1320 gtgtcgtgga actcaggcgc cctgaccagcggcgtgcaca ccttcccggc tgtcctacag 1380 tcctcaggac tctactccct cagcagcgtggtgaccgtgc cctccagcag cttgggcacc 1440 cagacctaca tctgcaacgt gaatcacaagcccagcaaca ccaaggtgga caagaaagtt 1500 gagcccaaat cttgtgacaa aactagctaattaatttaa 1539 5 22 PRT Artificial Sequence Synthetic 5 Asp Phe Gly ProAsp Trp Glu Asp Gly Asp Ser Tyr Asp Gly Ser Gly 1 5 10 15 Arg Gly PhePhe Asp Phe 20 6 17 PRT Artificial Sequence Synthetic 6 Gly Ile Ile ProIle Phe Gly Thr Ser Asn Tyr Ala Gln Lys Phe Arg 1 5 10 15 Gly 7 5 PRTArtificial Sequence Synthetic 7 Met Tyr Gly Phe Asn 1 5 8 20 PRTArtificial Sequence Synthetic 8 Gln Gln Tyr Gly Thr Ser Pro Tyr Thr PheGly Gln Gly Thr Lys Leu 1 5 10 15 Glu Ile Lys Arg 20 9 9 PRT ArtificialSequence Synthetic 9 Gly Ala Ser Thr Arg Ala Thr Gly Ile 1 5 10 13 PRTArtificial Sequence Synthetic 10 Arg Ala Ser Gln Ser Val Ser Ser Gly SerLeu Ala Trp 1 5 10 11 261 PRT Artificial Sequence Synthetic 11 Met LysTyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala 1 5 10 15 AlaGln Pro Ala Met Ala Glu Val Gln Leu Leu Glu Gln Ser Gly Ala 20 25 30 GluVal Lys Lys Pro Gly Ser Ser Val Gln Val Ser Cys Lys Ala Ser 35 40 45 GlyGly Thr Phe Ser Met Tyr Gly Phe Asn Trp Val Arg Gln Ala Pro 50 55 60 GlyHis Gly Leu Glu Trp Met Gly Gly Ile Ile Pro Ile Phe Gly Thr 65 70 75 80Ser Asn Tyr Ala Gln Lys Phe Arg Gly Arg Val Thr Phe Thr Ala Asp 85 90 95Gln Ala Thr Ser Thr Ala Tyr Met Glu Leu Thr Asn Leu Arg Ser Asp 100 105110 Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp Phe Gly Pro Asp Trp Glu 115120 125 Asp Gly Asp Ser Tyr Asp Gly Ser Gly Arg Gly Phe Phe Asp Phe Trp130 135 140 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Pro Gly Thr Lys GlyPro 145 150 155 160 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr SerGly Gly Thr 165 170 175 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe ProGlu Pro Val Thr 180 185 190 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser GlyVal His Thr Phe Pro 195 200 205 Ala Val Leu Gln Ser Ser Gly Leu Tyr SerLeu Ser Ser Val Val Thr 210 215 220 Val Pro Ser Ser Ser Leu Gly Thr GlnThr Tyr Ile Cys Asn Val Asn 225 230 235 240 His Lys Pro Ser Asn Thr LysVal Asp Lys Lys Val Glu Pro Lys Ser 245 250 255 Cys Asp Lys Thr Ser 26012 1539 DNA Artificial Sequence Synthetic 12 atgaaaaaga cagctatcgcgattgcagtg gcactggctg gtttcgctac cgtggcccag 60 gcggccgagc tcgtgttgacacagtctcca ggcaccctgt ctttgtctgc aggggaaaga 120 gccaccctct cctgcagggccagtcagagt gttagcagcg gctccttagc ctggtaccag 180 cagaaacctg gtcaggctcccaggctcctc atctacggtg catccaccag ggccactggc 240 atcccagaca ggttcagtggcagtgggtct gggacagact tcactctcac aatcggcaga 300 ctggagcctg aagatctcgcagtatattac tgtcagcagt atggtacctc accgtacact 360 tttggccagg ggaccaaactggagatcaaa cgaactgtgg ctgcaccatc tgtcttcatc 420 ttcccgccat ctgatgagcagttgaaatct ggaactgcct ctgttgtgtg cctgctgaat 480 aacttctatc ccagagaggccaaagtacag tggaaggtgg ataacgccct ccaatcgggt 540 aactcccagg agagcgtcacagagcatgac agcagggaca gcacctacag cctcggcagc 600 accctgacgc tgagcaaagcagactacgag aaacacaaag tctacgcctg cgaagtcacc 660 catcagggcc tgagttcgcccgtcacaaag agcttcaaca ggggagagtg ttaattctag 720 ataattaatt aggaggaatttaaaatgaaa tacctattgc ctacggcagc cgctggattg 780 ttattactcg ctgcccaaccagccatggcc gaggtgcagc tgctcgagca gtctggggct 840 gaggtgaaga agcctgggtcctcggtgcag gtctcctgca aggcctctgg aggcaccttc 900 agcatgtatg gtttcaactgggtgcgacag gcccctggac atggccttga gtggatggga 960 gggatcatcc ctatctttggtacatcaaac tacgcacaga agttccgggg cagagtcacg 1020 tttaccgcgg accaagccacgagcacagcc tacatggagc tgaccaacct gcgatctgac 1080 gacacggccg tctattattgtgcgagagat tttggccccg actgggaaga cggtgattcc 1140 tatgatggta gtggccgggggttctttgac ttctggggcc agggaaccct ggtcaccgtc 1200 tcctctcccg ggaccaagggcccatcggtc ttccccctgg caccctcctc caagagcacc 1260 tctgggggca cagcggccctgggctgcctg gtcaaggact acttccccga accggtgacg 1320 gtgtcgtgga actcaggcgccctgaccagc ggcgtgcaca ccttcccggc tgtcctacag 1380 tcctcaggac tctactccctcagcagcgtg gtgaccgtgc cctccagcag cttgggcacc 1440 cagacctaca tctgcaacgtgaatcacaag cccagcaaca ccaaggtgga caagaaagtt 1500 gagcccaaat cttgtgacaaaactagctaa ttaatttaa 1539 13 5 PRT Artificial Sequence Synthetic 13 GlyGly Gly Gly Ser 1 5 14 133 PRT Artificial Sequence Synthetic 14 Met AlaVal Gln Leu Leu Glu Gln Ser Gly Ala Glu Val Lys Lys Pro 1 5 10 15 GlySer Ser Val Gln Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser 20 25 30 MetTyr Gly Phe Asn Trp Val Arg Gln Ala Pro Gly His Gly Leu Glu 35 40 45 TrpMet Gly Gly Ile Ile Pro Ile Phe Gly Thr Ser Asn Tyr Ala Gln 50 55 60 LysPhe Arg Gly Arg Val Thr Phe Thr Ala Asp Gln Ala Thr Ser Thr 65 70 75 80Ala Tyr Met Glu Leu Thr Asn Leu Arg Ser Asp Asp Thr Ala Val Tyr 85 90 95Tyr Cys Ala Arg Asp Phe Gly Pro Asp Trp Glu Asp Gly Asp Ser Tyr 100 105110 Asp Gly Ser Gly Arg Gly Phe Phe Asp Phe Trp Gly Gln Gly Thr Leu 115120 125 Val Thr Val Ser Ser 130 15 110 PRT Artificial Sequence Synthetic15 Asp Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Ala Gly 1 510 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Gly 2025 30 Ser Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 3540 45 Ile Tyr Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 5055 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Gly Arg Leu Glu 6570 75 80 Pro Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Thr Ser Pro85 90 95 Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr 100 105110 16 20 PRT Artificial Sequence Synthetic 16 Gly Gly Gly Gly Ser GlyGly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser 20 1730 PRT Artificial Sequence Synthetic 17 Gly Gly Gly Gly Ser Gly Gly GlyGly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser Gly Gly GlyGly Ser Gly Gly Gly Gly Ser 20 25 30 18 40 PRT Artificial SequenceSynthetic 18 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly SerGly 1 5 10 15 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly SerGly Gly 20 25 30 Gly Gly Ser Gly Gly Gly Gly Ser 35 40 19 202 PRTArtificial Sequence Synthetic 19 Met Asn Arg Gly Val Pro Phe Arg His LeuLeu Leu Val Leu Gln Leu 1 5 10 15 Ala Leu Leu Pro Ala Ala Thr Gln GlyLys Lys Val Val Leu Gly Lys 20 25 30 Lys Gly Asp Thr Val Glu Leu Thr CysThr Ala Ser Gln Lys Lys Ser 35 40 45 Ile Gln Phe His Trp Lys Asn Ser AsnGln Ile Lys Ile Leu Gly Asn 50 55 60 Gln Gly Ser Phe Leu Thr Lys Gly ProSer Lys Leu Asn Asp Arg Ala 65 70 75 80 Asp Ser Arg Arg Ser Leu Trp AspGln Gly Asn Phe Pro Leu Ile Ile 85 90 95 Lys Asn Leu Lys Ile Glu Asp SerAsp Thr Tyr Ile Cys Glu Val Glu 100 105 110 Asp Gln Lys Glu Glu Val GlnLeu Leu Val Phe Gly Leu Thr Ala Asn 115 120 125 Ser Asp Thr His Leu LeuGln Gly Gln Ser Leu Thr Leu Thr Leu Glu 130 135 140 Ser Pro Pro Gly SerSer Pro Ser Val Gln Cys Arg Ser Pro Arg Gly 145 150 155 160 Lys Asn IleGln Gly Gly Lys Thr Leu Ser Val Ser Gln Leu Glu Leu 165 170 175 Gln AspSer Gly Thr Trp Thr Cys Thr Val Leu Gln Asn Gln Lys Lys 180 185 190 ValGlu Phe Lys Ile Asp Ile Val Val Leu 195 200 20 36 PRT ArtificialSequence Synthetic 20 Tyr Thr Ser Leu Ile His Ser Leu Ile Glu Glu SerGln Asn Gln Gln 1 5 10 15 Glu Lys Asn Glu Gln Glu Leu Leu Glu Leu AspLys Trp Ala Ser Leu 20 25 30 Trp Asn Trp Phe 35 21 15 PRT ArtificialSequence Synthetic 21 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly GlyGly Gly Ser 1 5 10 15

1. An isolated antibody or antibody fragment that specifically binds a CD4-inducible epitope on Human Immunodeficiency Virus (HIV) Env, wherein exposure of the CD4-inducible epitope is further enhanced by the binding of Env to a co-receptor for HIV, wherein the CD4-inducible epitope is distinct from the HIV co-receptor binding site on gp120.
 2. The isolated antibody or antibody fragment of claim 1, wherein the antibody or antibody fragment is selected by virtue of its ability to specifically bind to a CD4-inducible epitope on HIV Env that is enhanced by binding a co-receptor for HIV.
 3. An isolated antibody or antibody fragment, wherein the isolated antibody or antibody fragment is selected by virtue of its ability to specifically bind to a CD4-inducible epitope on Human Immunodeficiency Virus (HIV) Env that is enhanced by the binding of Env to a co-receptor for HIV, wherein the CD4-inducible epitope is distinct from the HIV co-receptor binding site on gp120.
 4. The isolated antibody or antibody fragment of claim 1, wherein the epitope is on gp120.
 5. The isolated antibody or antibody fragment of claim 1, wherein the epitope is on gp41.
 6. The isolated antibody or antibody fragment of claim 1, wherein the eipitope is on gp120-gp41.
 7. An isolated antibody or antibody fragment, wherein the isolated antibody or antibody fragment is selected by virtue of its ability to specifically bind to a complex comprising HIV gp120, CD4, and a co-receptor for HIV.
 8. The isolated antibody or antibody fragment of claim 7, wherein the complex further comprises gp41.
 9. The isolated antibody or antibody fragment of claim 1, wherein the HIV co-receptor is CCR5 or CXCR4.
 10. The isolated antibody or antibody fragment of claim 1, wherein the HIV gp120 epitope is an HIV-1 gp120 epitope.
 11. The isolated antibody or antibody fragment of claim 1, wherein the isolated antibody or antibody fragment has broadly neutralizing activity against HIV-1.
 12. The isolated antibody or antibody fragment of claim 1, wherein the isolated antibody or antibody fragment is monoclonal.
 13. The isolated antibody or active fragment thereof of claim 1, wherein the isolated antibody or antibody fragment is human or humanized.
 14. The isolated antibody or antibody fragment of claim 1, wherein the isolated antibody or antibody fragment is isolated from a phage display library.
 15. The isolated antibody or antibody fragment of claim 1, wherein the antibody or antibody fragment comprises the heavy chain of antibody Fab fragment X5 (SEQ ID NO: 3).
 16. The isolated antibody or antibody fragment of claim 1, wherein the antibody or antibody fragment comprises the light chain of antibody Fab fragment X5 (SEQ ID NO: 2).
 17. The isolated antibody or antibody fragment of claim 1, wherein the antibody or antibody fragment comprises the heavy chain of antibody Fab fragment X5 (SEQ ID NO: 3) and the light chain of antibody Fab fragment X5 (SEQ ID NO: 2).
 18. The isolated antibody or antibody fragment of claim 1, wherein the isolated antibody or antibody fragment comprises the CDR3 region (SEQ ID NO: 5) of the heavy chain of antibody Fab fragment X5.
 19. The isolated antibody or antibody fragment of claim 1, wherein the isolated antibody or antibody fragment comprises the CDR3 region (SEQ ID NO: 8) of the light chain of antibody Fab fragment X5.
 20. The isolated antibody or antibody fragment of claim 1, wherein a polypeptide comprising a heavy chain or light chain of the antibody or antibody fragment comprises soluble CD4.
 21. The isolated antibody or antibody fragment of claim 20, wherein a polypeptide comprising the heavy chain or light chain of the antibody or antibody fragment further comprises peptide T20.
 22. An isolated polypeptide comprising the heavy chain of antibody Fab fragment X5 (SEQ ID NO: 3).
 23. An isolated polypeptide comprising the light chain of antibody Fab fragment X5 (SEQ ID NO: 2).
 24. An isolated polypeptide comprising the CDR3 region (SEQ ID NO: 5) of the heavy chain of antibody Fab fragment X5.
 25. An isolated polypeptide comprising the CDR3 region (SEQ ID NO: 8) of the light chain of antibody Fab fragment X5.
 26. An isolated nucleic acid that encodes SEQ ID NO:
 3. 27. An isolated nucleic acid that encodes SEQ ID NO:
 2. 28. An isolated nucleic acid that encodes SEQ ID NO:
 5. 29. An isolated nucleic acid that encodes SEQ ID NO:
 8. 30. An isolated nucleic acid that encodes an antibody or antibody fragment comprising the heavy chain of antibody Fab fragment X5 (SEQ ID NO: 3) and the light chain of antibody Fab fragment X5 (SEQ ID NO: 2).
 31. The isolated nucleic acid of claim 30, wherein the isolated nucleic acid comprises the nucleotide sequence set forth in SEQ ID NO:
 4. 32. A vector comprising the isolated nucleic acid of claim
 26. 33. A cell comprising the vector of claim
 32. 34. A pharmaceutical composition comprising the isolated antibody or antibody fragment of claim 1, and a pharmaceutically acceptable carrier.
 35. The pharmaceutical composition of claim 34, further comprising soluble CD4.
 36. A pharmaceutical composition comprising a nucleic acid that encodes the isolated antibody or antibody fragment of claim 1, and a pharmaceutically acceptable carrier.
 37. The pharmaceutical composition of claim 36, wherein the nucleic acid is within an expression vector.
 38. A method of selecting an antibody or antibody fragment with broadly neutralizing activity against HIV, comprising detecting an antibody or antibody fragment that specifically binds a CD4-inducible epitope on HIV Env that is enhanced by the binding of Env to a co-receptor for HIV, wherein the CD4-inducible epitope is distinct from the HIV co-receptor binding site on gp120.
 39. The method of claim 37, wherein the antibody is exposed to a complex comprising HIV gp120, CD4, and a co-receptor for HIV.
 40. An antibody produced by the method of claim
 39. 41. A method of inhibiting entry of HIV into a cell, comprising administering to the cell an effective amount of an isolated antibody or antibody fragment that specifically binds a CD4-inducible epitope on HIV Env that is enhanced by the binding of Env to a co-receptor for HIV, wherein the CD4-inducible epitope is distinct from the HIV co-receptor binding site on gp120, thereby inhibiting entry of HIV into the cell.
 42. The method of claim 41, wherein the cell is in a mammal that is susceptible to infection by HIV and wherein the isolated antibody or antibody fragment is administered to the mammal.
 43. The method of claim 41, wherein the isolated antibody or antibody fragment is administered to the mammal by administering a nucleic acid encoding the isolated antibody or antibody fragment to the mammal.
 44. The method of claim 42, wherein the mammal is a primate.
 45. The method of claim 44, wherein the primate is a human.
 46. The method of claim 41, wherein the HIV is HIV-1.
 47. A method of inhibiting replication of HIV in a mammal that is susceptible to HIV infection, comprising administering to the mammal an effective amount of an isolated antibody or antibody fragment that specifically binds a CD4-inducible epitope on HIV Env that is enhanced by the binding of Env to a co-receptor for HIV, wherein the CD4-inducible epitope is distinct from the HIV co-receptor binding site on gp120, thereby inhibiting replication of HIV in the mammal.
 48. The method of claim 47, wherein the isolated antibody or antibody fragment is administered to the mammal by administering a nucleic acid encoding the isolated antibody or antibody fragment to the mammal.
 49. The method of claim 47, wherein the mammal is a primate.
 50. The method of claim 49, wherein the primate is a human.
 51. The method of claim 47, wherein the HIV is HIV-1.
 52. The method of claim 38, wherein the co-receptor is CCR5.
 53. The method of claim 38, wherein the co-receptor is CXCR4. 